U.S. patent application number 11/147714 was filed with the patent office on 2006-03-23 for method for detection of the presence or absence of methylthioadenosine phosphorylase (mtase) in a cell sample by detection of the presence or absence of mtase encoding nucleic acid in the cell sample.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Dennis A. Carson, Tsutomu Nobori, Kenji Takabayashi.
Application Number | 20060063172 11/147714 |
Document ID | / |
Family ID | 46277320 |
Filed Date | 2006-03-23 |
United States Patent
Application |
20060063172 |
Kind Code |
A1 |
Nobori; Tsutomu ; et
al. |
March 23, 2006 |
Method for detection of the presence or absence of
methylthioadenosine phosphorylase (MTAse) in a cell sample by
detection of the presence or absence of MTAse encoding nucleic acid
in the cell sample
Abstract
A method for detecting whether methyladenosine phosphatase
(MTAse) is present in a cell sample. In one respect, the method
comprises adding oligonucleotide probes to the sample, which probes
are capable of specifically hybridizing to any MTAse encoding
nucleic acid in the sample under conditions favoring that
hybridization. Absence of MTAse in a sample is considered to be
indicative of malignancy. Polynucleotides encoding MTAse, MTAse
peptides and antibodies to MTAse, as well as kits for performing
the methods of the invention, are provided.
Inventors: |
Nobori; Tsutomu; (Mie,
JP) ; Carson; Dennis A.; (Del Mar, CA) ;
Takabayashi; Kenji; (San Diego, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
The Regents of the University of
California
Oakland
CA
|
Family ID: |
46277320 |
Appl. No.: |
11/147714 |
Filed: |
June 7, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09780114 |
Feb 9, 2001 |
6911309 |
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11147714 |
Jun 7, 2005 |
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09072914 |
May 4, 1998 |
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09780114 |
Feb 9, 2001 |
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08827342 |
Mar 26, 1997 |
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09072914 |
May 4, 1998 |
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08459343 |
Jun 2, 1995 |
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08827342 |
Mar 26, 1997 |
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08176855 |
Dec 29, 1993 |
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08459343 |
Jun 2, 1995 |
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Current U.S.
Class: |
435/6.11 ;
435/6.12; 435/91.2; 536/23.2 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 1/25 20130101; C12N 9/1077 20130101; G01N 33/5005 20130101;
G01N 33/573 20130101; C07K 16/40 20130101 |
Class at
Publication: |
435/006 ;
435/091.2; 536/023.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C12P 19/34 20060101
C12P019/34 |
Claims
1. A method for detecting the presence of catalytically active and
catalytically inactive methylthioadenosine phosphorylase (MTAse) in
mammalian cells comprising (a) obtaining an assayable sample of
cells which are suspected of being MTAse deficient, (b) adding
oligonucleotide probes which will specifically hybridize to any of
the MTAse encoding nucleic acid present in the sample under
conditions which will allow the probes to detectably hybridize to
any such nucleic acid present in the sample, and (c) detecting
whether the MTAse encoding nucleic acid is present in the sample,
wherein the presence of said nucleic acid is indicative of the
presence of catalytically active or inactive MTAse in a cell.
2. A method according to claim 1 comprising further the step of
subjecting the sample to conditions favoring the selective
amplification of a nucleic acid which will encode for MTAse and
selectively amplifying any MTAse encoding nucleic acid present in
the sample.
3. A method according to claim 1 wherein the cells are derived from
a known malignancy.
4. A method according to claim 3 wherein the cells are also assayed
for MTAse catalytic activity.
5. A method according to claim 1 wherein the probes are derived
from the nucleotide sequence contained in SEQ. ID. No. 1.
6. A method according to claim 2 wherein the conditions employed
comprise a polymerase chain reaction.
7. An isolated polynucleotide which encodes MTAse having the
nucleic acid sequence shown in the Sequence Listing appended hereto
as SEQ. ID. No. 1.
8. A polynucleotide according to claim 7 having a nucleotide
sequence substantially similar to the sequence contained in SEQ. ID
No. 1.
9. A recombinant expression vector containing the polynucleotide of
claim 7.
10. Methylthioadenosine phosphorylase (MTAse) encoded by the
nucleic acid whose nucleotide sequence is set forth in SEQ. ID. No.
1, wherein the nucleic acid is expressed by a recombinant
expression vector.
11. A recombinant expression vector containing peptide encoding
fragments of the polynucleotide of claim 7.
12. MTAse peptides expressed by the recombinant expression vector
of claim 11.
13. Antibodies produced through immunization of an animal with the
MTAse peptides of claim 12.
14. Antibodies according to claim 13 wherein the antibodies are
monoclonal antibodies produced by hybridomas formed from cells of
the immunized animals.
15. Synthetic MTAse or MTAse peptide fragments.
16. Antibodies produced through immunization of an animal with the
MTAse or MTAse peptide fragments of claim 15.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This is a continuation-in-part of U.S. patent application
Ser. No. 08/459,343, filed on Jun. 2, 1995, which is in turn a
divisional application based on U.S. patent application Ser. No.
08/176,855, filed on Dec. 29, 1993.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to a method to detect
methylthioadenosine phosphorylase deficiency in mammalian cells, a
condition which is indicative of malignancy in those cells.
Detection of cells which are deficient in this enzyme allows those
cells to be targeted in chemotherapy to exploit the inability of
the cells to convert methylthioadenosine to methionine.
[0004] 2. History of the Invention
[0005] The amino acid methionine (MET) is necessary for the growth
of normal and malignant cells. In certain malignant cells this
requirement is absolute, i.e., without an adequate supply of MET,
the cells die.
[0006] In mammalian cells, MET is obtained from three sources. It
can be obtained in the diet, or through biochemical synthesis of
MET from L-homocysteine (homocysteine) or methylthioadenosine (MTA)
(a product of the polyamine biosynthetic pathway). In the latter
case, MTA is converted to MET by methylthioadenosine phosphorylase
(MTAse; EC 2.4.2.28).
[0007] In the past decade, researchers have identified many
malignant cell lines which lack MTAse and cannot, therefore,
convert MTA to MET. For example, Katamari, et al., Proc. Nat'l
Acad. Sci. USA, 78: 1219-1223 (1981) reported that 23% of 3 human
malignant tumor cell lines lacked detectable MTAse, while MTAse
activity was present in each of 16 non-malignant cell lines
studied. MTAse deficiency has also been reported as a
characteristic of non-small cell lung cancers (se, Nobori, et al.,
Cancer Res. 53:1098-1101 (1991)), in 6 lines of lymphoma and
leukemia cells (id.), in brain tumor cell lines and primary brain
tumor tissue samples (id.), and in other malignancies (see e.g.,
Kries, et al., Cancer Res. 33:1866-1869 (1973), Kries, et al.,
Cancer Trmt. Rpts. 63:1069-1072 (1979), and Rangione, et al.,
Biochem. J 281:533-538 (1992)). MTAse negative cells principally
fulfill their requirement for MET through conversion of
homocysteine. However, when homocysteine is not available, the
cells will generally die.
[0008] L-methionine-L-deamino-y-mercaptomethane lyase (ED 4.4.1.11;
METase) is known to degrade not only MET but also homocysteine.
Theoretically, therefore, one could starve malignant cells which
lack MTAse (i.e., MTAse negative cells) by degrading plasma MET and
homocysteine with METase. Normal MTAse positive cells would be
expected to fulfill their requirement for MET by the continued
conversion of MTA to MET.
[0009] One obstacle to the development of a successful approach to
MET starvation of malignant cells has been the need to identify
which malignancies are suitable targets for the therapy; i.e.,
which malignancies are MTAse negative. To that end, an assay was
developed which predicts whether a malignancy is MTAse negative by
determining whether any catalytic activity is present is a cell
culture (Seidenfeld, et al., Biochem. Biophays. Res. Commun.,
95:1861-1866, 1980). However, because of the commercial
unavailability of the radiochemical substrate required for the
assay, its use in routine evaluations is not presently feasible.
Moreover, the assay does not account for the catalytic lability of
MTAse in vitro by detecting whether any of the enzyme is present in
the cell culture regardless of whether it is catalytically active
at the time that the assay is performed.
[0010] This limitation of the activity assay could be avoided by
the development of an immunoassay which is sufficiently sensitive
to detect relatively minute quantities of enzyme. However, the
purification of the MTAse enzyme from natural sources to develop
antibodies for use in immunological detection of MTAse has proven
to be a laborious process which produces relatively poor yields
(Rangione, et al., J. Biol. Chem., 261:12324-12329, 1986).
[0011] The lack of a simple, efficient means of identifying MTAse
deficient cells has contributed in part to the continued
unavailability of an effective therapeutic approach to selective in
vivo MET starvation of MTAse deficient malignant cells. The present
invention addresses this need by providing a method for detection
of the presence or absence in a sample of the gene which encodes
for MTAse and by providing a recombinant source of MTAse.
BRIEF SUMMARY OF THE INVENTION
[0012] It is the object of the present invention to provide a
method for the detection of MTAse deficient cells (which will be
considered to be those cells in which the MTAse protein is not
detectably present in either a catalytically active or
catalytically inactive form). The method of the invention is based
on the assumption that MTEAse deficiency is due to deletion of the
gene which would encode for MTAse from the genome of the mammal
which has a MTAse negative malignancy. The method of the invention
is therefore directed to the detection of a polynucleotide inside
the MTAse protein coding domain of the mammal's genome which, if
present, would encode for MTAse but, if absent, would result in the
development of MTAse deficient cells.
[0013] More specifically, the present invention provides an assay
for detecting MTAse which includes the following steps: [0014] (a)
obtaining an assayable sample from the malignancy, [0015] (b)
subjecting the sample to conditions favoring the selective
amplification of a nucleic acid which will encode for MTAse, [0016]
(c) adding oligonucleotide probes which will specifically hybridize
to a nucleic acid which will encode for MTAse to the sample under
conditions which will allow the probes to detectably hybridize to
any such nucleic acid present in the sample, and [0017] (d)
detecting whether the nucleic acid is present in the sample.
[0018] Another aspect of the invention comprises a recombinant
MTAse obtained from the expression of MTAse by a suitable vector
from a polynucleotide which encodes MTAse. The availability of a
recombinant MTAse enables the production of highly pure material
with greater ease and in greater quantities than were obtainable
using the Rangione method (described supra) for the isolation and
purification of native MTAse.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 maps the genomic sequence for the gene for MTAse, and
indicates the location of exons in the polynucleotide. Presumed
exons are underlined; presumed introns are indicated by one or more
"N" substitutions for bases in the polynucleotide sequence. The
sequence depicted in FIG. 1 corresponds to the sequence contained
in SEQ. ID. No. 1 appended hereto.
DETAILED DESCRIPTION OF THE INVENTION
[0020] A. Method for Amplification of Any MTAse Present in a Cell
Sample
[0021] As noted above, it is an assumption of the invention that
MTAse deficiency in cells is the result of the deletion of the gene
from a mammal's genome which would normally encode for MTAse.
Because the invention is directed toward detecting the presence or
absence of this gene in a sample of cells which are suspected of
being MTAse negative, nucleic acids in the sample will preferably
be amplified to enhance the sensitivity of the detection method.
This amplification is preferably accomplished through the use of
the polymerase chain reaction (PCR), although the use of a chain
reaction in the polymerization step is not absolutely
necessary.
[0022] For use in the methods of the invention, a biological sample
is obtained which is suspected of containing MTAse deficient cells.
For example, the sample may comprise body fluid or cells, e.g.,
from a cell line, tissue or tumor. Such samples are obtained using
methods known in the clinical art, e.g. tumor cells may be acquired
by biopsy or surgical resection. Preferably, the cells are
essentially free from "contaminants"; i.e., cells, proteins and
similar components which are likely to falsify the result of the
method of the invention. For example, where solid tumors are used
as the source for genomic MTAse DNA, normal non-malignant cells and
MTAse which may be released from those cells during the procedure
performed to obtain the biological sample would be considered to be
contaminants.
[0023] The nucleic acid to be amplified in the sample will consist
of genomic or wild-type DNA which would normally be expected to
contain MTAse. This DNA (hereafter the "target DNA") to be
amplified is obtainable from a eukaryote, preferably a mammalian
organism. Most preferably, the genomic DNA is obtained from a
human.
[0024] Genomic DNA is isolated according to methods known in the
art, e.g., the method described by Maniatis, et al. (Molecular
Cloning, A Laboratory Manual, Cold Spring Habor Laboratory, 1982).
A working example demonstrating the isolation of a genomic clone of
human MTAse is provided herein wherein a cosmid gene library is
screened using an MTAse cDNA gene probe which is described further
below. However, those skilled in the art will recognize that other
suitable means of obtaining the DNA of the invention can be
used.
[0025] A full-length nucleotide sequence of the genomic clone for
MTAse is provided in the Sequence Listing appended hereto as SEQ.
ID. No. 1; exons in that sequence are depicted in the map shown in
FIG. 1. A strain of E. Coli containing the full-length genomic DNA
for rat MTAse has been deposited with the American Type Culture
Collection, Rockville, Md. by mail before Dec. 29, 1993 and
accepted on Dec. 30, 1993, under, collectively, Designation Nos.
55536, 55537, 55538, 55539 and 55540, which are E. coli
collectively transformed with the single full-length genomic clone
of rat MTAse described in SEQ. ID. No. 1. The host for each deposit
is E. coli. No admission that this deposit is necessary to enable
one to practice the invention is made or intended. The deposit
will, however, be maintained in viable form for whatever period is
or may be required by the patent laws applicable to this
disclosure.
[0026] Once the genomic DNA is obtained, the sample containing it
is subjected to conditions favoring the selective amplification of
the target nucleic acid. Preferably, the target nucleic acid will
be a polynucleotide portion of the gene which encodes MTAse (i.e.,
the "target polynucleotide"). The preferred means of amplifying the
target polynucleotide is by PCR. PCR is an in vitro method for the
enzymatic synthesis of specific DNA or RNA sequences using
oligonucleotide primers that hybridize to specific nucleic acid
sequences and flank the region of interest in target nucleic acid.
A repetitive series of cycles of template denaturation, primer
annealing and enzymatic extension of the annealed primers results
in an exponential accumulation of a specific nucleic acid fragment
defined at its termini by the 5' ends of the primers. The resulting
products (PCR products) synthesized in one cycle act as templates
for the next; consequently, the number of target nucleic acid
copies approximately doubles in every cycle.
[0027] The basic PCR techniques are described in U.S. Pat. Nos.
4,683,195 and 4,683,202 to Mullis, et al., the disclosures of which
are incorporated herein as examples of the conventional techniques
for performance of the PCR. However, the invention is not intended
to be limited to the use of the PCR techniques which are taught in
the '202 patent to Mullis, et al. Since the development of the
Mullis, et al. technique, many PCR based assays have been developed
which utilize modifications of that technique. These modifications
are well-known in the art and will not, therefore, be described in
detail here. However, for the purpose of illustrating the scope of
the art in this field, several of these modifications are described
as follows.
[0028] A PCR technique which provides an internal amplification
standard using a competitor template which differs from the target
nucleic acid in sequence and size is described in Proc. Natl. Acad.
Sci. USA (1990) 87:2725-2729 (Gilliland, et al., authors). Another
technique for performing "competitive" PCR which utilizes templates
which differ in sequence but not in size is described in Nuc.
Acids. Res., 21:3469-3472, (1993), (Kohsaka, et al., authors). This
technique is a particularly preferred technique for its use of
enzyme-linked immunoabsorbent assay (ELISA) technology to analyze
the amplified nucleic acid(s). A noncompetitive PCR technique which
utilizes site-specific oligonucleotides to detect mutations or
polymorphisims in genes which may also be applied to the method of
the invention is described in Proc. Natl. Acad. Sci. USA (1989)
86:6230-6234 (Saiki, et al., authors). Each of these techniques has
the advantage of utilizing hybridization probes which assist in
eliminating false positive results derived from any nonspecific
amplification which may occur during the PCR.
[0029] For further background, those skilled in the art may wish to
refer to Innis, et al., "Optimization of PCR's", PCR Protocols: A
Guide to Methods and Applications (Acad. Press, 1990). This
publication summarizes techniques to influence the specificity,
fidelity and yield of the desired PCR products.
[0030] Oliaonucleotide primers (at least one primer pair) are
selected which will specifically hybridize to a small stretch of
base pairs on either side (i.e., 5' and 3') of the MTAse target
polynucleotide (i.e., "flanking sequences"). Those skilled in the
art will readily be able to select suitable primers without undue
experimentation based on the polynucleotide sequence information
set forth in the Sequence Listing appended hereto as SEQ. ID. No. 1
and in FIG. 1.
[0031] For primer design, it is important that the primers do not
contain complementary bases such that they could hybridize with
themselves. To eliminate amplification of any contaminating
material which may be present in the sample, primers are preferably
designed to span exons (which, for the MTAse gene, are shown in
FIG. 1).
[0032] As noted above, it may not be necessary to utilize the chain
reaction in this polymerization step in order to adequately amplify
the nucleic acids in the sample. For example, where the technique
described by Kohsaka, et al., supra is utilized so the
polymerization step is performed on solid phase support means and
is followed by hybridization with target polynucleotide specific
probes, the sensitivity of the assay will be such that a single
polymerization of the target polynucleotide may be all that is
necessary.
[0033] Once the amplification step is complete, the PCR products
are assayed to determine thereby whether the gene to encode NVTAse
is present in the sample. Preferably, the double-stranded PCR
products will be bound to the solid phase so their strands may be
separated by denaturation, thereby allowing sequence-specific
probes to hybridize to the bound antisense strand of the PCR
product to detect the gene substantially as described in Kohsaka,
et al., supra. Alteratively, the PCR products will be removed from
the reaction environment and separated from the amplification
mixture prior to the addition of probes for hybridization to the
double-stranded PCR products. In this latter approach, the PCR
products are separated from the amplification mixture according to
methods known in the art with regard to the particular method
chosen for detection; e.g., by gel exclusion, electrophoresis or
affinity chromatography.
[0034] Detection of the amplified product may be achieved by using
hybridization probes which are stably associated with a detectable
label. A label is a substance which can be covalently attached to
or firmly associated with a nucleic acid probe which will result in
the ability to detect the probe. For example, a level may be a
radioisotope, an enzyme substrate or inhibitor, an enzyme, a
radiopaque substance (including colloidal metals), a fluorescors, a
chemiluminescent molecule, liposomes containing any of the above
labels, or a specific binding pair member. A suitable label will
not lose the quality responsible for detectability during
amplification.
[0035] Those skilled in the diagnostic art will be familiar with
suitable detectable labels for use in in vitro detection assays.
For example, suitable radioisotopes for in vitro use include
.sup.3H, .sup.125I, .sup.131I, .sup.32P, .sup.14C, .sup.35S.
Amplified fragments labeled by means of a radioisotope may be
detected directly by gamma counter or by densitometry of
autoradiographs, by Southern blotting of the amplified fragments
combined with densitometry. Examples of suitable chemiluminescent
molecules are acridines or luminol. Target sequences hybridized
with probes derivatized with acridium ester are protected from
hydrolysis by intercalation. Examples of suitable fluorescers are
fluorescein, phycobiliprotein, rare earth chelates, dansyl or
rhodamine.
[0036] Examples of suitable enzyme substrates or inhibitors are
compounds which will specifically bind to horseradish peroxidase,
glucose oxidase, glucose-6-phosphate dehydrogenase,
.beta.-galactosidase, pyruvate kinase or alkaline phosphatase
acetylcholinesterase. Examples of radiopaque substance are
colloidal gold or magnetic particles.
[0037] A specific binding pair comprises two different molecules,
wherein one of the molecules has an area on its surface or in a
cavity which specifically binds to a particular spatial and polar
organization of another molecule. The members of the specific
binding pair are often referred to as a ligand and receptor or
ligand and anti-ligand. For example, if the receptor is an antibody
the ligand is the corresponding antigen. Other specific binding
pairs include hormone-receptor pairs, enzyme substrate pairs,
biotin-avidin pairs and glycoprotein-receptor pairs. Included are
fragments and portions of specific binding pairs which retain
binding specificity, such a fragments of immunoglobulins, including
Fab fragments and the like. The antibodies can be either monoclonal
or polyclonal. If a member of a specific binding pair is used as a
label, the preferred separation procedure will involve affinity
chromatography.
[0038] If no amplified product can be detected in the assay
described above, this is indicative of MTAse deficiency in the
cells present in the sample. Because normal (i.e., nonmalignant)
cells will always be expected to have MTAse present in detectable
quantities, the finding of MTAse deficiency indicates that the
analyzed genomic DNA was obtained from malignant cells. The assay
of the invention is particularly suitable for diagnostic purposes,
e.g. for the diagnosis of MTAse deficiency associated with
neoplasms, particularly malignant neoplasms.
[0039] Where desired, the sample can be prescreened for MTAse
catalytic activity using the method described by Seidenfeld, et
al., Biochem. Biophys. Res. Commun., 95:1861-1866 (1980); see also,
Example I, infra). The inventive assay will then be used to
determine whether the gene encoding MTAse is present in cells in
the sample. The sample may also be tested for the presence of
catalytically active or inactive protein for the purpose of
screening out contaminants; i.e., nonmalignant cells in the sample.
A suitable immunoassay for use in this regard is described in
Nobori, et al., Cancer Res. 53:1098-1101 (1991) and in co-pending
U.S. patent application Ser. No. 80/176,413, filed on Dec. 29,
1993.
[0040] B. Production of Synthetic or Recombinant MTAse
Polynucleotides and Peptides
[0041] It is another object of the present invention to provide
polynucleotides (in particular, oligonucleotides) which enable the
amplification of a MTAse specific nucleic acid sequence. The
strategy for designing such oligonucleotides will consider the
aspects mentioned above. Such oligonucleotides are particularly
useful for diagnosis of MTAse deficiency associated with
malignancy.
[0042] The invention also provides synthetic and recombinant MTAse
and MTAse peptides as well as polynucleotides which encode MTAse
and MTAse peptides. As used herein, "polynucleotide" refers to a
polymer of deoxyribonucleotides or ribonucleotides, in the form of
a separate fragment or as a component of a larger construct. DNA
encoding MTAse or an MTAse peptide of the invention can be
assembled from cDNA fragments or from oligonucleotides which
provide a synthetic gene which is capable of being expressed in a
recombinant transcriptional unit. Polynucleotide sequences of the
invention include DNA, RNA and cDNA sequences. A polynucleotide
sequence can be deduced from the genetic code, however, the
degeneracy of the code must be taken into account. Polynucleotides
of the invention include sequences which are degenerate as a result
of the genetic code.
[0043] Peptides and polynucleotides of the invention include
functional derivatives of MTAse, MTAse peptides, and nucleotides
encoding therefor. By "functional derivative" is meant the
"fragments," "variants," "analogs," or "chemical derivatives" of a
molecule. A "fragment" of a molecule, such as any of the
polynucleotides of the present invention, includes any nucleotide
subset of the molecule. A "variant" of such molecule refers to a
naturally occurring molecule substantially similar to either the
entire molecule, or a fragment thereof. An "analog" of a molecule
refers to a non-natural molecule substantially similar to either
the entire molecule or a fragment thereof.
[0044] A molecule is said to be "substantially similar" to another
molecule if the sequence of amino acids in or, in the case of
polynucleotides, produced by both molecules is substantially the
same. Substantially similar amino acid molecules will possess a
similar biological activity. Thus, provided that two molecules
possess a similar activity, they are considered variants as that
term is used herein even if one of the molecules contains
additional amino acid residues not found in the other, or if the
sequence of amino acid residues is not identical.
[0045] As used herein, a molecule is said to be a "chemical
derivative" of another molecule when it contains additional
chemical moieties not normally a part of the molecule. Such
moieties may improve the molecule's solubility, absorption,
biological half life, etc. The moieties may alternatively decrease
the toxicity of the molecule, eliminate or attenuate any
undesirable side effect of the molecule, etc. Moieties capable of
mediating such effects are disclosed, for example, in Remington's
Pharmaceutical Sciences, 16th Ed., Mack Publishing Co., Easton, Pa.
(1980).
[0046] Minor modifications of the MTAse primary amino acid sequence
may result in proteins which have substantially equivalent activity
as compared to the MTAse enzyme and peptides described herein. Such
modifications may be deliberate, as by site-directed mutagenesis,
or may be spontaneous. All of the proteins and peptides produced by
these modifications are included herein as long as the biological
activity of MTAse still exists. Further, deletion of one or more
amino acids can also result in a modification of the structure of
the resultant molecule without significantly altering its
biological activity. This can lead to the development of a smaller
active molecule which would have broader utility. For example, one
can remove amino or carboxy terminal amino acids which may not be
required for the enzyme to exert the desired catalytic or antigenic
activity.
[0047] The term "conservative variation" as used herein denotes the
replacement of an amino acid residue by another, biologically
similar residue. Examples of conservative variations include the
substitution of one hydrophobic residue such as isoleucine, valine,
leucine or methionine for another, or the substitution of one polar
residue for another, such as the substitution of arginine for
lysine, glutamic for aspartic acids, or glutamine for asparagine,
and the like. The term "conservative variation" also includes the
use of a substituted amino acid in place of an unsubstituted parent
amino acid provided that the antibodies raised to the substituted
polypeptide also immunoreact with the unsubstituted
polypeptide.
[0048] DNA sequences for use in producing MTAse and MTAse peptides
of the invention can also be obtained by several methods. For
example, the DNA can be isolated using hybridization procedures
which are well known in the art. These include, but are not limited
to: 1) hybridization of probes to genomic or cDNA libraries to
detect shared nucleotide sequences; 2) antibody screening of
expression libraries to detect shared structural features and 3)
synthesis by the polymerase chain reaction (PCR).
[0049] Hybridization procedures are useful for the screening of
recombinant clones by using labeled mixed synthetic oligonucleotide
probes where each probe is potentially the complete complement of a
specific DNA sequence in the hybridization sample which includes a
heterogeneous mixture of denatured double-stranded DNA. For such
screening, hybridization is preferably performed on either
single-stranded DNA or denatured double-stranded DNA. Hybridization
is particularly useful in the detection of cDNA clones derived from
sources where an extremely low amount of MRNA sequences relating to
the polypeptide of interest are present. In other words, by using
stringent hybridization conditions directed to avoid non-specific
binding, it is possible, for example, to allow the autoradiographic
visualization of a specific CDNA clone by the hybridization of the
target DNA to that single probe in the mixture.
[0050] An MTAse containing cDNA library can be screened by
injecting the various mRNA derived from cDNAs into oocytes,
allowing sufficient time for expression of the cDNA gene products
to occur, and testing for the presence of the desired cDNA
expression product, for example, by using antibody specific for
MTAse or by using probes for the repeat motifs and a tissue
expression pattern characteristic of MTAse. Alternatively, a cDNA
library can be screened indirectly for MTAse peptides having at
least one epitope using antibodies specific for the polypeptides.
As described in Section C below, such antibodies can be either
polyclonally or monoclonally derived and used to detect expression
product indicative of the presence of MTAse cDNA.
[0051] Screening procedures which rely on nucleic acid
hybridization make it possible to isolate any gene sequence from
any organism, provided the appropriate probe is available.
Oligonucleotide probes, which correspond to a part of the sequence
encoding the protein in question, can be synthesized chemically.
This requires that short, oligopeptide stretches of amino acid
sequence must be known. The DNA sequence encoding the protein can
be deduced from the genetic code, however, the degeneracy of the
code must be taken into account. It is possible to perform a mixed
addition reaction when the sequence is degenerate. This includes a
heterogeneous mixture of denatured double-stranded DNA. For such
screening, hybridization is preferably performed on either
single-stranded DNA or denatured double-stranded DNA.
[0052] The development of specific DNA sequences encoding MTAse or
fragments thereof can also be obtained by: 1) isolation of
double-stranded DNA sequences from the genomic DNA: 2) chemical
manufacture of a DNA sequence to provide the necessary codons for
the polypeptide of interest; and 3) in vitro synthesis of a
double-stranded DNA sequence by reverse transcription of mRNA
isolated from a eukaryotic donor cell. In the latter case, a
double-stranded DNA-complement of mRNA is eventually formed which
is generally referred to as cDNA.
[0053] In the present invention, the polynucleotide and any
variants thereof encoding MTAse may be inserted into a recombinant
expression vector. The term "recombinant expression vector" refers
to a plasmid, virus or other vehicle known in the art that has been
manipulated by insertion or incorporation of the appropriate
genetic sequences. Such expression vectors contain a promoter
sequence which facilitates the efficient transcription of the
inserted genetic sequence of the host.
[0054] Transformation of a host cell with recombinant DNA may also
be carried out by conventional techniques as are well known to
those skilled in the art. Host cells may be eukaryotic (such as
Chinese hamster ovary cells) or prokaryotic (such as bacteria or
yeast). Where the host is prokaryotic, such as E. coli, competent
cells which are capable of DNA uptake can be prepared from cells
harvested after exponential growth phase and subsequently treated
by the CaCl.sub.2 method by procedures well known in the art.
Alternatively, MgCl.sub.2 or RbCl can be used. Transformation can
also be performed after forming a protoplasm to the host cell or by
electroporation.
[0055] Isolation and purification of microbially expressed MTAse,
or fragments thereof, provided by the invention, may be carried out
by those of ordinary skill in the art using conventional means
including preparative chromatography and immunological separations
involving monoclonal or polyclonal antibodies.
[0056] Based on the information contained in SEQ. ID. No. 1, the
full-length amino acid sequence for MTAse may be readily deduced.
Using this information, MTAse and MTAse peptides may also be
synthesized without undue experimentation by commonly used methods
such as t-BOC or FMOC protection of alpha-amino groups. Both
methods involve stepwise synthesis whereby a single amino acid is
added at each step starting from the C terminus of the peptide (see
Coligan, et al., Current Protocols in Immunology, Wiley
Interscience, 991, Unit 9). Peptides of the invention can also be
synthesized by various well known solid phase peptide synthesis
methods, such as those described by Merrifield, J. Am. Chem. Soc.,
85:2149 (1962), and Stewart and Young, Solid Phase Peptides
Synthesis, (Freeman, San Francisco, 27-62, 1969), using a
copoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g
polymer.
[0057] In this latter method, completion of chemical synthesis, the
peptides can be deprotected and cleaved from the polymer by
treatment with liquid HF-10% anisole for about 1/4-1 hours at
0.degree. C. After evaporation of the reagents, the peptides are
extracted from the polymer with 1% acetic acid solution which is
then lyophilized to yield the crude material. This can normally be
purified by such techniques as gel filtration on Sephadex G-15
using 5% acetic acid as a solvent. Lyophilization of appropriate
fractions of the column will yield the homogeneous peptide or
peptide derivatives, which can then be characterized by such
standard techniques as amino acid analysis, thin layer
chromatography, high performance liquid chromatography, ultraviolet
absorption spectroscopy, molar rotation, solubility, and
quantitated by the solid phase Edman degradation.
[0058] C. Production of Anti-MTAse Antibodies
[0059] The antigenicity of MTAse peptides can be determined by
conventional techniques to determine the magnitude of the antibody
response of an animal which has been immunized with the peptide.
Generally, the MTAse peptides which are used to raise the
anti-MTAse antibodies should generally be those which induce
production of high titers of antibody with relatively high affinity
for MTAse. Such peptides may be purified for use as immunogens
using, for example, the method described in Rangione, et al., (J.
Biol. Chem., supra) or the methods for obtaining MTAse peptides
described above.
[0060] Once antigenic peptides are prepared, antibodies to the
immunizing peptide are produced by introducing peptide into a
mammal (such as a rabbit, mouse or rat). For purposes of
illustration, the amino acid sequences of two antigenic MTAse
peptides are provided in the Sequence Listing appended hereto as
SEQ ID. Nos. 2 and 3. Antibodies produced by rabbits immunized with
these peptides showed a 50% maximal response to purified MTAse at,
respectively, a 1:1500 and a 1:4000 dilution.
[0061] A multiple injection immunization protocol is preferred for
use in immunizing animals with the antigenic MTAse peptides (see,
e.g., Langone, et al., eds., "Production of Antisera with Small
Doses of Immunogen: Multiple Intradermal Injections", Methods of
Enymology (Acad. Press, 1981). For example, a good antibody
response can be obtained in rabbits by intradermal injection of 1
mg of the antigenic MTAse peptide emulsified in Complete Freund's
Adjuvant followed several weeks later by one or more boosts of the
same antigen in Incomplete Freund's Adjuvant.
[0062] If desired, the immunizing peptide may be coupled to a
carrier protein by conjugation using techniques which are
well-known in the art. Such commonly used carriers which are
chemically coupled to the peptide include keyhole limpet hemocyanin
(KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus
toxoid. The coupled peptide is then used to immunize the animal
(e.g. a mouse or a rabbit). Because MTAse is presently believed to
be conserved among mammalian species, use of a carrier protein to
enhance the immunogenecity of MTAse proteins is preferred.
[0063] Polyclonal antibodies produced by the animals can be further
purified, for example, by binding to and elution from a matrix to
which the peptide to which the antibodies were raised is bound.
Those of skill in the art will know of various techniques common in
the immunology arts for purification and/or concentration of
polyclonal antibodies, as well as monoclonal antibodies (see, for
example, Coligan, et al., Unit 9, Current Protocols in Immunology,
Wiley Interscience, 1991).
[0064] For preparation of monoclonal antibodies, immunization of a
mouse or rat is preferred. The term "antibody" as used in this
invention is meant also to include intact molecules as well as
fragments thereof, such as for example, Fab and F(ab').sub.2. which
are capable of binding the epitopic determinant. Also, in this
context, the term "mAb's of the invention" refers to monoclonal
antibodies with specificity for MTAse.
[0065] The general method used for production of hybridomas
secreting monoclonal antibodies ("mAb's"), is well known (Kohler
and Milstein, Nature, 256:495, 1975). Briefly, as described by
Kohler and Milstein the technique comprised lymphocytes isolated
from regional draining lymph nodes of five separate cancer patients
with either melanoma, teratocarcinoma or cancer of the cervix,
glioma or lung, were obtained from surgical specimens, pooled, and
then fused with SHFP-1. Hybridomas were screened for production of
antibody which bound to cancer cell lines.
[0066] Confirmation of MTAse specificity among mab's can be
accomplished using relatively routine screening techniques (such as
the enzyme-linked immunosorvent assay, or "ELISA") to determine the
elementary reaction pattern of the mAb of interest.
[0067] It is also possible to evaluate an mAb to determine whether
it has the same specificity as a mAb of the invention without undue
experimentation by determining whether the mAb being tested
prevents a mAb of the invention from binding to MTAse isolated as
described above. If the mAb being tested competes with the mAb of
the invention, as shown by a decrease in binding by the mAb of the
invention, then it is likely that the two monoclonal antibodies
bind to the same or a closely related epitope.
[0068] Still another way to determine whether a mAb has the
specificity of a mAb of the invention is to pre-incubate the mAb of
the invention with an antigen with which it is normally reactive,
and determine if the mAb being tested is inhibited in its ability
to bind the antigen. If the mAb being tested is inhibited then, in
all likelihood, it has the same, or a closely related, epitopic
specificity as the mAb of the invention.
[0069] D. MTAse Detection Kits
[0070] MTAse detection kits may be prepared for use in laboratory
and clinical settings which include reagents useful in the methods
described above. For example, a kit for use in the method of
Section A, supra, would preferably include oligonucleotide primers
(produced as described in Section B above), detectably labelled
hybridization probes and reagant coated microtiter plates. The kit
could also include the antibodies described in Section C above for
use in immunological detection of MTAse protein (as described in
co-pending application Ser. No. 08/176,413, filed Dec. 29,
1993).
[0071] The invention having been fully described, examples
illustrating its practice are provided below. These examples should
be considered as exemplars only and not as limiting the scope of
the invention.
[0072] In the Examples, the following abbreviations are use:
AS=anti-sense, DTT=dithiothreitol; min=minutes;
MTAse=5'-deoxy-5'-methylt-hioadenosine phosphorylase;
PCR=polymerase chain reaction; S=sense; SSc=0.3 M NaCl, 0.03 M
sodium citrate dihydrate; v/v=volume per volume; SDS=sodiumdodecyl
sulfate.
EXAMPLE I
[0073] Test for MTAse Catalytic Activity in a Sample
[0074] The phosphorolysis activity of MTAse was determined by
measuring the formation of [methyl-.sup.14C]
5-methylthioribose-1-phosphate from [methyl-.sup.14C]
5'-deoxy-5'-methylthioadenosine (Seidenfeld et al., Biochem.
Biophys. Res. Commun. 95, 1861-1866, 1980). In a total volume of
200 microliters the standard reaction mixture contained 50 mM
potassium phosphate buffer, pH 7.4, 0.5 mM [methyl-.sup.14C]
5'deoxy-5'-methylthioadenosine (2.sup.5.times.10 CPM/mmol), 1 mM
DTT and the indicated amounts of enzyme. After incubation at
37.degree. C. for 20 min, the reaction was stopped by addition of
50 microliters of 3 M trichloroacetic acid and 200 microliter
aliquots were applied to a 0.6.times.2 cm column of "DOWEX"
50-H.TM. equilibrated with water. The [methyl-.sup.14C] 5
methylthioribose-1-phosphate was eluted directly into scintillation
vials containing 2 ml of -0.1 M Hcl.
EXAMPLE II
[0075] Purification of Native MTAse from Rat Liver
[0076] MTAse was isolated from rat liver modifying the method of
Rangione et al. (J. Biol. Chem. 261, 12324-12329, 1986). 50 g of
fresh rat liver were homogenized in a Waring Blendor with 4 volumes
of 10 mM potassium phosphate buffer, pH 7.4, containing 1 mM DTT
(Buffer A). The homogenate was centrifuged (1 h at 15,000.times.g),
and the resulting supernatant was subjected to ammonium sulfate
fractionation. The precipitate between 55 and 75% saturation was
collected by centrifugation (15,000.times.g for 20 min) and
dissolved in a minimal volume of Buffer A. The sample was then
dialyzed overnight against three changes of 100 volumes of the same
buffer.
[0077] The sample was clarified by centrifugation at 15,000.times.g
for 30 min and then applied to a DEAE-Sephacryl column
(1.5.times.18 cm; Pharmacia) previously equilibrated with Buffer A.
After washing with 80 ml of equilibration buffer, a linear gradient
(80 ml) or 0-0.3 M NaCl in buffer A was applied. MTAse activity was
eluted between 0.1 and 0.15 M NaCl. Fractions containing at least
0.06 units/mg of protein were concentrated 20-fold by
ultrafiltration (Amicon PM-10 Diaflow membranes) and dialyzed
extensively against 25 mM potassium phosphate buffer, pH 7.4
containing 1 mM DTT (Buffer B). The sample was then applied to a
hydroxyapatite column (1.times.12 cm) (Bio-Rad). After elution of
non-absorbed proteins with Buffer B, the column was washed with
about 40 ml of 50 mM potassium phosphate buffer, pH 7.4, containing
1 mM DTT.
[0078] MTAse was then eluted using a linear gradient (40 ml) of
50-250 mM potassium phosphate, pH 7.4. Fractions containing MTAse
activity were concentrated 30-fold by ultrafiltration and freed
from dithiothreitol by repeated concentration and dilution with 50
mM potassium phosphate buffer, ph 7.4. The partially purified
enzyme was then applied to a column (0.8.times.3 cm) of
organomercurial agarose (Bio-Rad) equilibrated with 50 Mm phosphate
buffer, pH 7.4. Elution of the column was carried out stepwise with
a) 50 mM potassium phosphate buffer, pH 7.4; b) 50 mM potassium
phosphate buffer, pH 7.4, 2 M KCl; and c) 50 mM potassium phosphate
buffer, pH 7.4, 2 M KCl, 40 mM 2-mercaptoethanol. The enzyme was
then eluted with 50 mM potassium phosphate buffer, pH 7.4, 2 M KCl,
200 mM 2-mercaptoethanol. Fractions containing at least 3 units/mg
of protein were pooled, concentrated to 1 ml by ultrafiltration,
and dialyzed overnight against 1000 volumes of 10 mM Tris/HCl, pH
7.4, 1 M DTT (Buffer C). As a final purification step, aliquots of
the sample (1 ml) were injected at a flow rate of 1 ml/min into a
"MONO Q" column (Pharmacia) pre-equilibrated with 10 mM Tris/HCl,
pH 7.4, containing 1 mM DTT, and 0.5 ml fractions were collected.
MTAse activity was eluted between 0.08 and 0.14 M NaCl in Buffer C.
The fractions were concentrated to 0.5 ml by ultrafiltration and
dialyzed against 1000 volumes of Buffer B.
EXAMPLE III
[0079] Determination of a Partial Amino Acid Sequence for Rat
MTAse
[0080] The purified sample was lyophilized, dissolved in a 50
microliter sample loading buffer (1% sodium dodecylsulfate (SDS),
10% glycerin, 0.1 M DTT and 0.001% bromphenolblue) and loaded onto
a 0.5 mm thick 10% SDS polyacrylamide gel (Bio Rad "MINIGEL"
apparatus). After electrophoresis, proteins were electroblotted for
2 hr onto nitrocellulose (0.45 millimeter pore size, Millipore) in
a Bio-Rad transblot system using transfer buffer (15 mM Tris, 192
mM glycine and 20% methanol, pH 8.3) as described by Towbin, et al.
(Proc. Nat'l Acad. Sci. USA 76, 4340-4345, 1979).
[0081] After transfer, proteins were reversibly stained with
Ponceau S.TM. (Sigma) using a modification of the method described
by Salinovich and Montelaro (Anal. Biochem. 156, 341-347, 1987).
The nitrocellulose filter was immersed for 60 sec in a solution of
0.1% Ponseau S dye in 1% aqueous acetic acid. Excess stain was
removed from the blot by gentle agitation for 1-2 min in 1% aqueous
acetic acid. The protein-containing region detected by stain was
cut out, transferred to an Eppendorf tube (1.5 ml), washed with
distilled water, and incubated for 30 min at 37.degree. C. in 1.2
ml of 0.5% polyvinyl-pyrrolidone (average molecular weight=40,000;
PVP-40, Sigma) dissolved in 100 mM acetic acid in order to prevent
absorption of the protease to the nitrocellulose during digestion.
Excess PVP-40 was removed by extensive washing with water (at least
five changes).
[0082] Nitrocellulose strips were then cut in small pieces of
approximately 1 mm.times.1 mm and put back into the same tube. The
protein on the nitrocellulose pieces was digested as described
before (Los et al., Science 243:217-220, 1989). Trypsin (10 pmol)
in 100 microliter of 100 mM Tris-HCl, pH 8.2/acetonitrile, 95;5
(v/v) is added and incubated at 37.degree. C. overnight. After
digestion, peptide-containing supernatant was acidified with 30
microliter of 10% trifluoroacetic acid, moved quickly on a Vortex,
and centrifuged at 15,000.times.g for 1 min. The supernatant was
removed and immediately injected into a reverse-phase HPLC system
(Beckrnann) equipped with a Brownlee Aquapore Bu-300 analytical
column (2.1.times.100 mm).
[0083] Eluent D 0.1% trifluoroacetic acid (sequenal grade, in
water) was pumped through the column for 5 min at a flow rate of
200 microliter/min before the flow was reduced to 100
microliter/min and the gradient is started with Eluent E
(0.08-0.095% trifluoroacetic acid in acetonitrile/H.sub.2O, 70;30
(v/v). Based on UV absorption at 215 nm peptide-containing
fractions were collected manually into Eppendorf tubes.
Representative fractions 60 and 77 were subjected to amino acid
sequencing (ABI 477A Protein Sequencer with 120A Online PTH-AA
Analyzer). Thus independent partial amino acid sequences of rat
MTAse were obtained.
EXAMPLE IV
[0084] Amplification of a DNA Fragment Encoding Part of the Human
MTAse Gene
[0085] Two sets of oligonucleotide primers with different
polarities were synthesized. Each oligonucleotide was designed to
include a unique restriction site at its 5'-end (EcoRI or BamHI) in
order to facilitate the subsequent cloning of the amplified DNA
fragment. For use in PCR amplification total cDNA was isolated from
1 million plaque-forming-units (pfu) of human placenta cDNA gene
library (Clontech) using the "Lambda-TRAP" kit (Clontech). The PCR
reaction was carried out in a total volume of 100 microliters
containing 1 microgram of total cDNA from human placenta cDNA gene
library, 1.times.PCR buffer (10 mM KCl, 10 mM Tris-HCl, pH 8.3, 2.5
mM MgCl.sub.2), 0.2 mM of each dNTP, 100 mg each of sense and
anti-sense primers and 10 units of Taq DNA polymerase, Stoffel
Fragment ("AMPLI TAQ", Perkin-Elmer Cetus).
[0086] Forty cycles were performed with the "GENE AMP" PCR System
9600 (Perkin-Elmer Cetus), each cycle consisting of denaturation
(92.degree. C., 1 min), annealing (55.degree. C., 2 min) and
extension (72.degree. C., 2 min). The PCR product was separated
electrophoretically on a 0.8% agarose gel in 1.times.TA buffer (40
mM Tris-acetate, 20 mM Na-acetate, 2 mM EDTA, pH 7.9) and a 450 bp
DNA fragment was amplified. The PCR amplification product was
double digested with restriction enzymes EcoRL/BamHI, separated on
a 0.8% agarose gel in 1.times.TA buffer, recovered from the gel
using "GENE CLEAN".TM. Kit (Bio101), subcloned into EcoRI/BamHI cut
pBluescript vector SK.sup.+ (Stratagene) and sequenced by the
dideoxytermination method using universal sequencing primer
("SEQUENASE".TM. Version 1.0 DNA sequencing kit, USB).
EXAMPLE V
[0087] Screening of a Human Placenta cDNA Gene Library
[0088] Sequence analysis of the PCR amplified product (Example IV)
shows perfect coincidence with the C-terminal amino acid sequence
of peptide 1 (SEQ. ID No. 5). Using the 450 bp DNA fragment as
hybridization probe, a human placenta cDNA gene library (Clontech)
was screened. To that end, E. coli strain Y1090 host cells were
incubated overnight with vigorous shaking at 37.degree. C. in LB
medium (per liter: 10 g trypcone, 5 g yeast extract, 10 g NaCl)
containing 0.2% maltose and 10 mM MgSP. For each culture plate, 0.3
ml of host cell culture was mixed with 3.times.10 sup.4 pfu phage
and incubated for 20 min at 37.degree. C. The mixtures of host
cells and phage were added to 8 ml of LB-medium containing 0.7%
agarose (LB-top-agarose) that were pre-warmed at 48.degree. C. and
poured onto 20 agar plates (135.times.15 mm). Plaques were visible
after incubation for 6 to 8 h at 37.degree. C. and plates were
chilled to 4.degree. C. for 1 h. Plaques were transferred to
Colony/Plaque Screen nylon transfer membranes (NEN Research
Products, Dupont Boston, Mass.) for 3 min, followed by denaturation
(2.times. in 0.5 N NaOH for 2 min), renaturation (2 times in 1.0 M
Tris-HCl, pH7.5 for 2 min) and fixation by air drying.
Prehybridization of 20 membranes was carried out in two plastic
bags containing 10 membranes each, using 20 ml of prehybridization
buffer (1% SDS, 2.times.SSC, 10% dextran sulphate, 50% deionized
formamide) for 4 h at 42.degree. C.
[0089] The 450 bp EcoRI-BamHI fragment of the partial human MTAse
gene was labeled with [alpha-.sub.32P]dATP (3,000 Ci/mmol) using a
nicktranslation kit (Boehringer Mannheim), separated from
unincorporated radioactivity on a NICK-column (Pharmacia),
denatured by heating at 96.degree. C. for 10 min, chilled on ice
and added to the membranes in the plastic bags with the probe
concentration being 106 dpm/ml. The specific activity of the
labeled probe is around 10.sup.8 dpm/microgram. Hybridization was
performed overnight at 42.degree. C. After hybridization, membranes
were washed at room temperature three times for 5 min with excess
of 2.times.SSC, then at 65.degree. C. for 20 min with 2.times.SSC,
0.1% SDS and once at room temperature for 20 min with
0.2.times.SSC, 0.1% SDS. The washed membranes were exposed to an
X-ray film overnight.
[0090] The agar plugs containing several plaques around a positive
signal were removed into a 1 ml sterile phage diluent (50 mM
Tris-HCl, pH 7.5, 0.1 M NaCl, 8 mM MgSO.sub.4, 0.01% gelatine) and
rescreened as above mentioned, until the pure positive plaques were
obtained. From screening of approximately half a million plaques, 6
independent positive clones were obtained. After amplification on
LB plates, each phage DNA of positive clones was purified using a
"Lambda-TRAP" kit (Clontech). Purified phage DNAs were cut with
EcoRI enzyme to obtain the whole insert, but because of the
existence of an EcoRI site inside of the insert, two bands were cut
out from all the clones.
[0091] Two EcoRI insert fragments (850 bp and 1100 bp) from the
representative phage clone, designated as MTAp-1, were subcloned
into EcoRI-cut pBluescript SK.sup.+ vector (Stratagene). These
subclones were designated MTAP-2 (850 bp) and MTAP-3 (1100 bp),
respectively. Restriction analysis and DNA sequencing of these two
subclones reveal that subclone MTAP-2 has an open reading frame
coding for 254 amino acids comprising the amino acid sequence
corresponding to peptide 3 at its C-terminus (homology 90%).
Calculated from the molecular weight of human MTAse of 32 kDa (F.
D. Rangione et al., J. Biol. Chem. 261:12324-12329, 1986), it
covers over 85% of total protein. About 50 amino acids (at least
150 bp on DNA level) are missing.
EXAMPLE VI
[0092] Primer Extension to Obtain the Missing 5' End cDNA of
MTAse
[0093] To obtain the 5'-terminal missing DNA fragment, RACE (rapid
amplification of cDNA ends) was applied (Loh et al., Science
243:217-220, 1989; Frohman, et al. PNAS 85:8998-9002, 1988). One
microgram of poly (A+) RNA from human placenta (Clontech) in 6.25
microliters of H.sub.2O was heated at 65.degree. C. for 5 min,
quenched on ice, and added to 4 microliters of 5.times.RTC buffer
(250 mM Tris-HCl, pH 8.15, 30 mM MgCl.sub.2, 200 mM KCl, 5 mM DTT),
4 microliters (0.4 mg/ml) of actinomycin D (Boehringer), 1
microliters of each dNTP (20 mM), 0.25 microliters (10 units) of
RNasin (Boehringer), 1 microliter of [alpha-.sup.35S]dATP (1443
Ci/mmol), 1 microliter of human MTAse specific anti-sense
oligonucleotide 3 AS and 10 units of avian myeloblastosis virus
reverse transcriptase (Boehringer). The mixture was incubated for 2
hr at 42.degree. C.
[0094] Excess primer and dNTPs were removed as follows; the 20
microliter cDNA pool was applied to a NICK-column (Pharmacia) and
two-drop fractions were collected. Fractions 5-8 relative to the
first peak of radioactivity were pooled, precipitated with 1/10
volume of 7.5 M NHOAc and 2.5 volume of ethanol at -80.degree. C.
for 2 hr, centrifuged at 15,000.times.g for 30 min at 4.degree. C.,
washed with 0.5 ml of 80% ethanol, dried under reduced pressure
(Speedvac) and dissolved in 20 microliter of H.sub.2O. For tailing,
1.5 microliter of dGTP (20 mM), 2.4 microliter of CoCl1.sub.2 (25
mM), 6 microliter of 5.times. tailing buffer (1 mM potassium
cacodylate, 125 mM Tris-HCl, pH 6.6, 1.25 mg/ml bovine serum
albumin) and 0.5 microliter of (15 units) terminal deoxynucleotidyl
transferase (Boehringer) were added.
[0095] The mixture was incubated for 1 hr at 37.degree. C., heated
for 15 min at 65.degree. C., extracted once with the same volume of
TE-buffer (10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA) saturated with
phenol, and then precipitated with ethanol as mentioned above. The
tailed cDNA pool was dissolved in 20 microliter of H.sup.o and 1
microliter was used for PCR. For amplification two additional
primers were synthesized. One primer was a MTAse specific
anti-sense primer which locates 180 bp upstream of the position of
oligonucleotide 3AS. The other was a primer for the poly(G) end.
Amplification was performed for 40 cycles as described above. Each
cycle consisted of denaturation (92.degree. C., 1 min), annealing
(50.degree. C., 2 min) and extension (72.degree. C., 0.5 min).
[0096] The PCR product was separated electrophoretically on a 0.8%
agarose gel. The obtained 520 bp DNA fragment was specifically
amplified. After purification on a 0.8% agarose preparative gel,
the 520 bp DNA fragment was digested with Not I and Bcl I (the
relevant restriction sites being present in the overlapping domain
between the extended DNA fragment and the original fragment of
subclone MTAP-2) and subcloned into Not I/BamHI-cut pBluescript
SK.sup.+ vector (Stratagene). Sequence analysis of three
independent subclones, designated MTAP-4, MTAP-5 and MTAP-6,
respectively, revealed that each of these clones contains an
exactly matched amino acid sequence in the overlapping domain.
[0097] The lengths of these three primer-extended cDNA subclones
are slightly different. This implies that they are independent PCR
products and that their sequences reflect the correct mRNA sequence
without any base mid-incorporation during PCR amplification. The
combination of the new upstream sequence with the start codon ATG
(coding for methionine) and the down-stream sequence from subclone
MTAP-2 generates an open reading frame coding for 283 amino
acids.
EXAMPLE VII
[0098] Expression of Recombinant Human MTAse in E. Coli
[0099] The full-length cDNA of human MTAse was constructed by
adding the primer-extended cDNA fragment of subclone MTAP-4, which
contains the largest insert of the three subclones obtained in
Example VI, to the 5'end of the DNA insert of subclone MTAP-2 using
their common restriction site HindIII. The Not I/HindIII-DNA
fragment from subclone MTAP-4 and the large HindIII/EcoRI fragment
from subclone MTAP-2 were mixed and subcloned into Not I/EcoRI-cut
pBluescript vector SK+ (Stratagene). The obtained subclone
containing a full-length cDNA of human MTAse was designated MTAP-7.
To check the authenticity of this cDNA clone, the recombinant
protein was expressed using E. coli expression vector pKK223-3
equipped with the Taq promotor (Pharmacia).
[0100] To generate a new site EcoRI-site at the 5'end and a Pst I
site at 3'-end of the cDNA fragment, PCR was used applying a
5'-primer oligonucleotide comprising the Shine-Dalgamo (SD)
sequence and another 3'-primer. Amplification was performed for 20
cycles as mentioned above with each cycle consisting of
denaturation (92.degree. C., 1 min), annealing (55.degree. C., 1
min) and extension (72.degree. C., 1 min). The PCR product was
digested with restriction enzymes EcoRI/Pst I, purified
electrophoretically on a 0.8% agarose gel and subcloned into
EcoRI/PstI-cut pBluescript vector SK.sup.+ (Stratagene).
[0101] After checking the full sequence of the insert in the
subclone referred to as NITAP-8, the EcoRI/Pst I fragment was cut
out and subcloned into EcoRI/Pst I cut pKK223-3 vector yielding
human MTAse cDNA in an E. coli expression vector. The subclone
designated as MTAP-9 was transformed into E. coli strain JM105. The
enzymatic activity and the spectrum of total proteins of
transformed cells with and without
isopropyl-beta-D-thiogalactopyranoside (IPTG) induction were
analyzed. A singe transformed colony was inoculated into 2 ml of LB
medium and incubated overnight at 37.degree. C., 20 microliter of
this overnight culture are added into two plastic tubes, each
containing fresh 2 ml of LB medium (1/100 dilution).
[0102] After incubation at 37.degree. C. for 1 hr to one tube 20
microliter of 0.1 M IPTG added for induction to give a final
concentration of 1 mM IPTG and incubated at 37.degree. C. for
additional 4 hr. After harvesting the cells by centrifugation at
15,000.times.g for 5 min, the cells were resuspended in 100
microliters of phosphate buffer (50 mM potassium phosphate, pH 7.5,
1 mM DTT), disrupted by sonication on ice at step 3 for 0.5 min and
crude cell extracts are obtained by centrifugation at
15,000.times.g for 10 min.
[0103] The protein concentration was determined using the Bradford
method (Bio-Rad, Protein Assay). The same amounts of samples with
and without IPTG induction were analyzed for enzymatic activity and
subjected to electrophoresis on a 10% SDS polyacrylamide gel. The
crude extract obtained from IPTG induced cells displayed an MTAse
activity which is more than 5-fold higher than that of non-induced
cells. Furthermore, on the SDS gel a new induced protein band (31
kDa) was detected.
EXAMPLE VIII
[0104] Cloning and Partial Characterization of the MTAse Genomic
Clone
[0105] For the most efficient amplification of DNA fragment by PCR
for diagnostic purposes, its size should preferably be less than
500 bp. The cDNA sequence reflects the sum of exons, which are
normally separated by introns which makes it difficult to find out
an adequate sequence with appropriate size from the cDNA sequence.
To overcome this problem, a genomic clone of human MTAse was
isolated. A cosmid gene library constructed from human placenta DNA
(Clontech) was screened using MTAse cDNA gene probe, the Not
I/EcoRI fragment from subclone MTAP-7. Transformed E. coli cells
from the library are plated on LB plates containing ampicillin (50
mg/l) with a colony density of 1-2.times.10.sup.4/135.times.15 mm
plate.
[0106] The following procedures were performed as described in
Example IV. From half a million colonies, a single positive colony
designated as MAP-10 was isolated and partially characterized by
PCR analysis and by direct sequencing. Two primers, a sense
oligonucleotide located 120 bp upstream of the stop codon and an
anti-sense oligonucleotide located 20 bp downstream of the stop
codon were synthesized and used for PCR analysis. PCR was performed
for 25 cycles, each cycle consisting of denaturation (92.degree.
C., 1 min), annealing (55.degree. C., 2 min) and extension
(72.degree. C., 5 min). The PCR products were separated on a 0.8%
agarose gel.
[0107] The location of exons in the MTAse gene using the
above-described technique is depicted in FIG. 1.
EXAMPLE IX
[0108] Application of MTAse Sequence-Specific Oligonucleotides to
Malignant Cell Lines to Detect the Presence or Absence of MTAse
Therein
[0109] To test the usefulness of oligonucleotides PCR was applied
for several cell lines which were known to contain MTAse positive
and negative cells. Genomic DNAs were isolated as described in
Example VIII and 1 microgram thereof was used for PCR.
Amplification was performed for 40 cycles as described above, with
each cycle consisting of denaturation (92.degree. C., 1 min),
annealing (55.degree. C., 1 min), and extension (72.degree. C., 1/2
min). The PCR products were analyzed on a 1.5% agarose gel. No
MTAse was detected in cell lines which were known to be MTAse
negative, while MTAse was detected in the MTAse positive cell
lines.
Sequence CWU 1
1
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