U.S. patent application number 11/588744 was filed with the patent office on 2007-03-01 for atp-binding cassette protein responsible for cytotoxin resistance.
This patent application is currently assigned to The Gov. of U.S.A. as Represented by the Secretary of the Department of Health and Human Services. Invention is credited to Rando L. Allikmets, Susan E. Bates, Michael Dean, Antonio T. Fojo.
Application Number | 20070048810 11/588744 |
Document ID | / |
Family ID | 37423198 |
Filed Date | 2007-03-01 |
United States Patent
Application |
20070048810 |
Kind Code |
A1 |
Dean; Michael ; et
al. |
March 1, 2007 |
ATP-binding cassette protein responsible for cytotoxin
resistance
Abstract
This invention provides for a novel ATP-binding cassette protein
which is responsible for cytotoxin resistance. The invention also
provides for methods of expressing the protein and assays for
identification of inhibitors of the protein.
Inventors: |
Dean; Michael; (Frederick,
MD) ; Allikmets; Rando L.; (Cornwall-on-the-Hudson,
NY) ; Bates; Susan E.; (Bethesda, MD) ; Fojo;
Antonio T.; (Rockville, MD) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
8TH FLOOR
SAN FRANCISCO
CA
94111
US
|
Assignee: |
The Gov. of U.S.A. as Represented
by the Secretary of the Department of Health and Human
Services
Rockville
MD
|
Family ID: |
37423198 |
Appl. No.: |
11/588744 |
Filed: |
October 26, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09856927 |
Sep 19, 2001 |
7138493 |
|
|
PCT/US99/28107 |
Nov 24, 1999 |
|
|
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11588744 |
Oct 26, 2006 |
|
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|
60110473 |
Nov 30, 1998 |
|
|
|
Current U.S.
Class: |
435/7.23 ;
435/320.1; 435/325; 435/69.1; 530/350; 530/388.8; 536/23.5 |
Current CPC
Class: |
C07K 14/705
20130101 |
Class at
Publication: |
435/007.23 ;
435/069.1; 435/320.1; 435/325; 530/350; 530/388.8; 536/023.5 |
International
Class: |
G01N 33/574 20060101
G01N033/574; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C07K 14/82 20070101 C07K014/82; C07K 16/30 20070101
C07K016/30 |
Claims
1-2. (canceled)
3. A eukaryote cell genetically altered to overexpress an
ATP-binding cassette protein having the following properties: i.
conferring mitoxantrone resistance on S1-M1-80 human colon
carcinoma cells when expressed in the cells; and, ii. specifically
binding to polyclonal antibodies which specifically bind to a
member of the group of proteins depicted in Seq. ID. No. 2 or ID.
No. 4.
4. A cell of claim 3, wherein the cell is genetically altered by
transformation of the cell with an exogenous DNA comprising an
expression cassette encoding the ATP-binding cassette protein.
5. A cell of claim 4, wherein the expression cassette comprises a
heterologous promoter operatively linked to the DNA encoding the
ATP-binding cassette protein.
6. A cell of claim 3, wherein the cell has an endogenous copy of
the ATP-binding cassette protein and the genetic alteration
comprises insertion of DNA which can serve as an enhancing element
or as a second promoter where the insertion is upstream of the
endogenous promoter operatively linked to the ATP-binding cassette
protein and where the inserted DNA increases the basal expression
levels of ATP-binding cassette protein.
7. A DNA encoding a ATP-binding cassette protein wherein the
protein is characterized by having the following properties: i.
conferring mitoxantrone resistance on S1-M1-80 human colon
carcinoma cells when expressed in the cells; and, ii. specifically
binding to polyclonal antibodies which specifically bind to a
member of the group of proteins consisting of those depicted in
Seq. ID. No. 2 or ID. No. 4.
8. The DNA of claim 7, wherein the encoded protein has 95% identity
to the amino acids depicted in Seq. ID. No. 2 or ID. No. 4.
9. The DNA of claim 7, wherein the DNA encoding the protein has a
sequence identical to that depicted in Seq. ID. No. 1 or No. 3.
10. A process for over expressing ATP-binding cassette protein in a
cell comprising a first step of either: i. transforming the cell
with an expression cassette which directs the expression of
ATP-binding cassette protein; or, ii. selecting a cell having an
endogenous copy of the ATP-binding cassette protein, and
transforming the cell with DNA which can serve as an enhancing
element or as a second promoter where the insertion is upstream of
the endogenous promoter operatively linked to the ATP-binding
cassette protein and where the inserted DNA increases the basal
expression levels of ATP-binding cassette protein; and a second
step of, culturing the transformed cell under conditions where the
levels of ATP-binding cassette protein are increased above the
basal levels of the non-transformed cells with the proviso that the
ATP-binding cassette protein has the following properties: a.
confers mitoxantrone resistance on S1-M1-80 human colon carcinoma
cells when expressed in the cells; and, b. specifically binds to
polyclonal antibodies which specifically bind to a member of the
group of proteins depicted in Seq. ID. No. 2 or ID. No. 4.
11. The process of claim 10, wherein the ATP-binding cassette
protein has 95% homology to the amino acids depicted in Seq. ID.
No. 2 or ID. No. 4.
12. The process of claim 10, wherein the protein has the amino
acids depicted in Seq. ID. No. 2 or ID. No. 4.
13-20. (canceled)
Description
RELATED APPLICATIONS
[0001] The present application is a division of U.S. Ser. No.
09/856,927, which is the national phase under 35 U.S.C. .sctn.371
of PCT/US99/28107, filed Nov. 24, 1999, which claims priority to
U.S. Ser. No. 60/110,473, filed Nov. 30, 1998. The contents of each
of the above-mentioned documents are incorporated herein in the
entirety.
FIELD OF THE INVENTION
[0002] This invention provides for a novel ATP-binding cassette
protein which is responsible for cytotoxin resistance. The
invention also provides for methods of expressing the protein and
assays for identification of inhibitors of the protein.
BACKGROUND OF THE INVENTION
[0003] The multidrug resistance/ATP-binding cassette (MDR/ABC)
superfamily in humans includes genes whose products represent
membrane proteins involved in energy-dependent transport of a wide
variety of substrates across a membrane (see, e.g., Dean, M. and
Allikmets, R. (1995) Curr. Opin. Genet. Dev. 5, 79-785). The
overexpression of ABC transporters has been linked with drug
resistance since the 1976 discovery of P-glycoprotein and the
subsequent cloning of the encoding gene, MDR-1. Resistance ensues
from reduced intracellular drug concentrations, a result of active
drug efflux. The subsequent identification of the multidrug
resistance associated protein (MRP), encoded by the MRP gene,
heralded a new era that recognized the complexity of the problem
and catalyzed the search for additional transporters. MDR-1 and MRP
are members of the expanding superfamily of ATP-binding cassette
proteins (ABC proteins). This superfamily is comprised of a large
and diverse group of proteins that transport solutes across
biological membranes. Transmembrane domains are thought to form a
pathway through which substrates cross cell membranes, while two
ATP-binding domains hydrolyze ATP to accomplish substrate
transport. Mutations in ABC transporters have been identified as
etiologic in diseases including hyperinsulinemic hypoglycemia of
infancy, adrenoleukodystrophy, and cystic fibrosis. The
transporters MDR-1 and MRP, and possibly the multispecific organic
anion transporter, cMOAT, are thought to be involved in both normal
excretion of xenobiotics and in drug resistance. The ABC
superfamily also includes a number of transporters without known
function and the potential exists to identify additional
transporters which mediate drug resistance.
[0004] Recent studies have described a number of cell lines with
resistance to mitoxantrone that exhibit multidrug resistance
without overexpression of MRP. In addition to mitoxantrone, these
cell lines are particularly resistant to anthracyclines, and have
an energy-dependent reduction in the accumulation of daunomycin and
mitoxantrone. Cell lines possessing this phenotype include sublines
derived by selection of leukemic cells, as well as breast, colon,
and gastric carcinomas.
SUMMARY OF THE INVENTION
[0005] The present invention thus provides for the first time,
nucleic acids encoding a new transporter protein that mediates drug
resistance. These proteins are generically called ATP binding
cassette proteins (ABC proteins). The ABC protein of the invention
is referred to as MXR1. It is also known as ABCP, and is also known
as ABCG2.
[0006] In one aspect, the present invention provides an isolated
ATP-binding cassette protein that confers mitoxantrone resistance
to S1-M1-80 human colon carcinoma cells when expressed in the
cells; and specifically binds to polyclonal antibodies which
specifically bind to a member of the group of proteins depicted in
SEQ ID NO. 2 or SEQ ID. NO. 4; and has a molecular weight between
about 70 kDa and about 75 kDa.
[0007] In one embodiment, the MXR1 protein has the sequence
depicted in SEQ ID NO. 2 or SEQ ID NO. 4. In another embodiment,
the protein has 95% identity to the amino acids depicted in SEQ ID
NO. 2 or SEQ ID. NO. 4.
[0008] In another aspect, the present invention provides a
eukaryotic cell genetically altered to overexpress an ATP-binding
cassette protein that confers mitoxantrone resistance on S1-M1-80
human colon carcinoma cells when expressed in the cells; and
specifically binds to polyclonal antibodies which specifically bind
to a member of the group of proteins depicted in SEQ ID NO. 2 or
SEQ ID. NO. 4.
[0009] In one embodiment, the cells of the invention are
genetically altered by transformation of the cell with an exogenous
DNA comprising an expression cassette encoding the ATP-binding
cassette protein. In another embodiment, the expression cassette
also employs a heterologous promoter operatively linked to the DNA
encoding the ATP-binding cassette protein. In another embodiment,
the cell may have an endogenous copy of the ATP-binding cassette
protein with a genetic alteration comprising insertion of DNA that
serves as an enhancing element or as a second promoter where the
insertion is upstream of the endogenous promoter operatively linked
to the ATP-binding cassette protein and where the inserted DNA
increases the basal expression levels of ATP-binding cassette
protein.
[0010] In another aspect, the present invention provides for DNA
encoding an ATP-binding cassette protein wherein the protein
confers mitoxantrone resistance on S1-M1-80 human colon carcinoma
cells when expressed in the cells and specifically binds to
polyclonal antibodies which specifically bind to the proteins
depicted in SEQ ID NO. 2 or SEQ ID NO. 4.
[0011] In one embodiment, the DNA encodes for the protein of SEQ ID
NO. 2 or SEQ ID NO. 4 and in other embodiments the DNA encodes a
protein that has 95% identity to the amino acids depicted in SEQ ID
NO. 2 or SEQ ID NO. 4. In another embodiment, the DNA has the
sequence depicted in SEQ ID NO. 1 or SEQ ID NO. 3.
[0012] In another aspect, the present invention provides a process
for over expressing ATP-binding cassette protein in a cell
comprising a first step of either (1) transforming the cell with an
expression cassette which directs the expression of ATP-binding
cassette protein; or, (2) selecting a cell having an endogenous
copy of the ATP-binding cassette protein, and transforming the cell
with DNA which can serve as an enhancing element or as a second
promoter where the insertion is upstream of the endogenous promoter
operatively linked to the ATP-binding cassette protein and where
the inserted DNA increases the basal expression levels of
ATP-binding cassette protein; and a second step of culturing the
transformed cell under conditions where the levels of ATP-binding
cassette protein are increased above the basal levels of the
non-transformed cells. The ATP binding protein of this embodiment
is one that confers mitoxantrone resistance on S1-M1-80 human colon
carcinoma cells when expressed in the cells; and, specifically
binds to polyclonal antibodies which specifically bind to a member
of the group of proteins depicted in SEQ ID NO. 2 or SEQ ID No.
4.
[0013] In one embodiment, the ATP binding cassette protein has 95%
homology to the amino acids depicted in SEQ ID NO. 2 or SEQ ID No.
4. In yet another embodiment, the protein has the amino acids
depicted in SEQ ID NO. 2 or SEQ ID No. 4.
[0014] In another aspect, the present invention provides a method
of screening for inhibitors of cytotoxin resistance in cells. The
method comprises (1) culturing a cell genetically altered by the
introduction of heterologous DNA which permits the overexpression
an ATP-binding cassette protein that confers mitoxantrone
resistance on S1-M1-80 human colon carcinoma cells when expressed
in the cells; and specifically binds to polyclonal antibodies which
specifically bind to a member of the group of proteins depicted in
SEQ ID NO. 2 or SEQ ID NO. 4; and, (2) contacting the cell with a
cytotoxin in an amount that permits cell survival due to the
resistance conferred by the ATP-binding cassette protein; and, (3)
contacting the cell with a compound that inhibits the biological
activity of the ATP-binding cassette protein; and, (4) detecting
the inhibition by measuring growth inhibition of the cells.
[0015] In one embodiment, the cytotoxin is mitoxantrone. In another
embodiment, the cytotoxin is daunomycin. In another embodiment, the
cell is a carcinoma cell. In another embodiment, the ATP-binding
cassette protein has 95% homology to the amino acids depicted in
SEQ ID NO. 2 or SEQ ID NO. 4. In another embodiment, the ATP
binding cassette protein has the amino acid sequence depicted in
SEQ ID NO. 2 or SEQ ID NO. 4.
[0016] In another aspect, the invention provides a binding protein
that specifically binds to an ATP-binding cassette protein which
has 95% homology to the amino acids depicted in SEQ ID NO. 2 or SEQ
ID NO. 4. In one embodiment, the binding protein is an antibody,
and in another embodiment, the binding protein is a monoclonal
antibody.
Non-Competitive Assay Formats.
[0017] Immunoassays for detecting the ATP binding cassette protein
may be either competitive or noncompetitive. Noncompetitive
immunoassays are assays in which the amount of captured analyte (in
this case the protein) is directly measured. In one preferred
"sandwich" assay, for example, the capture agent (anti-ABC
antibodies) can be bound directly to a solid substrate where they
are immobilized. These immobilized antibodies then capture the ATP
binding cassette protein present in the test sample. The ATP
binding cassette protein thus immobilized is then bound by a
labeling agent, such as a second ATP binding cassette antibody
bearing a label. Alternatively, the second antibody may lack a
label, but it may, in turn, be bound by a labeled third antibody
specific to antibodies of the species from which the second
antibody is derived. The second can be modified with a detectable
moiety, such as biotin, to which a third labeled molecule can
specifically bind, such as enzyme-labeled streptavidin.
DETAILED DESCRIPTION
[0018] Introduction
[0019] The present invention provides for the first time, nucleic
acids encoding the ATP binding cassette (ABC) protein, MXR1. These
nucleic acids and the subunits they encode are part of a
superfamily of membrane proteins and a part of a multidrug
resistance subfamily, involved in energy dependent transport of
substrates across membranes. The gene which is the subject of the
present invention encodes a 658 amino acid protein that is highly
expressed in placenta, in fetal brain and liver, and in at least
two mitoxantrone resistant cancer cell lines. The strong expression
of the gene in the placenta indicates that the MXR1 protein is
important in the transfer of specific molecules in or out of the
placenta. The overexpression in the cancer cell lines indicates
that the protein is involved with multidrug resistance in cancer
cells.
[0020] The invention also provides an assay for screening for
inhibitors of cytotoxin resistance in cells. The assay involves
culturing a cell that has been genetically altered by the
introduction of heterologous DNA which permits the overexpression
of an ATP-binding cassette protein that confers mitoxantrone
resistance and contacting the cell with a cytotoxin and contacting
the cell with a compound that inhibits the biological activity of
the ATP-binding cassette protein and detecting the inhibition by
measuring growth inhibition of the cells.
[0021] Definitions
[0022] "ATP-binding cassette protein" refers to a protein having an
ATP-binding cassette (ABC) (see, e.g., Allikmets et al., Human
Molecular Genetics 5; 1649-1655 (1996)) which is involved in
transporting substrates across cell membranes. They are energy
dependent (ATP) and have defined transmembrane domains.
[0023] As used herein, an "antibody" refers to a protein
functionally defined as a binding protein and structurally defined
as comprising an amino acid sequence that is recognized by one of
skill as being derived from the framework region of an
immunoglobulin encoding gene of an animal producing antibodies. An
antibody can consist of one or more polypeptides substantially
encoded by immunoglobulin genes or fragments of immunoglobulin
genes. The recognized immunoglobulin genes include the kappa,
lambda, alpha, gamma, delta, epsilon and mu constant region genes,
as well as myriad immunoglobulin variable region genes. Light
chains are classified as either kappa or lambda. Heavy chains are
classified as gamma, mu, alpha, delta, or epsilon, which in turn
define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively.
[0024] A typical immunoglobulin (antibody) structural unit is known
to comprise a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (VL) and variable heavy chain (VH) refer to
these light and heavy chains respectively.
[0025] Antibodies exist as intact immunoglobulins or as a number of
well characterized fragments produced by digestion with various
peptidases. Thus, for example, pepsin digests an antibody below the
disulfide linkages in the hinge region to produce F(ab)'2, a dimer
of Fab which itself is a light chain joined to VH-CH1 by a
disulfide bond. The F(ab)'2 may be reduced under mild conditions to
break the disulfide linkage in the hinge region thereby converting
the F(ab')2 dimer into an Fab' monomer. The Fab' monomer is
essentially an Fab with part of the hinge region (see, Fundamental
Immunology, W. E. Paul, ed., Raven Press, N.Y. (1993), for a more
detailed description of other antibody fragments). While various
antibody fragments are defined in terms of the digestion of an
intact antibody, one of skill will appreciate that such Fab'
fragments may be synthesized de novo either chemically or by
utilizing recombinant DNA methodology. Thus, the term antibody, as
used herein also includes antibody fragments either produced by the
modification of whole antibodies or synthesized de novo using
recombinant DNA methodologies. Preferred antibodies include single
chain antibodies (antibodies that exist as a single polypeptide
chain), more preferably single chain Fv antibodies (sFv or scFv) in
which a variable heavy and a variable light chain are joined
together (directly or through a peptide linker) to form a
continuous polypeptide. The single chain Fv antibody is a
covalently linked VH-VL heterodimer which may be expressed from a
nucleic acid including VH- and VL-encoding sequences either joined
directly or joined by a peptide-encoding linker. Huston, et al.
(1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. While the VH and
VL are connected to each as a single polypeptide chain, the VH and
VL domains associate non-covalently. The first functional antibody
molecules to be expressed on the surface of filamentous phage were
single-chain Fv's (scFv); however, alternative expression
strategies have also been successful. For example Fab molecules can
be displayed on phage if one of the chains (heavy or light) is
fused to g3 capsid protein and the complementary chain exported to
the periplasm as a soluble molecule. The two chains can be encoded
on the same or on different replicons; the important point is that
the two antibody chains in each Fab molecule assemble
post-translationally and the dimer is incorporated into the phage
particle via linkage of one of the chains to g3p (see, e.g., U.S.
Pat. No. 5,733,743). The scFv antibodies and a number of other
structures converting the naturally aggregated, but chemically
separated light and heavy polypeptide chains from an antibody V
region into a molecule that folds into a three dimensional
structure substantially similar to the structure of an
antigen-binding site are known to those of skill in the art (see
e.g., U.S. Pat. Nos. 5,091,513, 5,132,405, and 4,956,778).
Particularly preferred antibodies include all those that have been
displayed on phage (e.g., scFv, Fv, Fab and disulfide linked Fv
(Reiter et al., (1995) Protein Eng. 8: 1323-1331). Antibodies can
also include diantibodies and miniantibodies.
[0026] An "anti-MXR1" or an "anti-ABC" antibody is an antibody or
antibody fragment that specifically binds an MXR1 protein or an
ATP-binding cassette protein respectively.
[0027] "Binding protein" is a general term for a protein that
specifically binds to a target ligand or cognate molecule. It
includes either member of a binding pair. It would also include
receptor-like molecules, hormones, antibodies, antigens, and
importantly proteins identified as selective or specific binders
from a randomized library of proteins displayed on phage.
[0028] "Endogenous" refers to a naturally occurring element of a
cell or organism that is naturally produced by the cell or organism
as part of its normal life cycle.
[0029] "Exogenous" refers to non-naturally occurring elements of a
cell which are introduced by the hand of man. Transformation of
cell with nucleic acid introduces exogenous DNA elements. An
exogenous DNA element usually denotes a nucleic acid that has been
isolated, cloned, and ligated to a nucleic acid with which it is
not combined in nature, and or introduced into and/or expressed in
a cell or cellular environment other than the cell or cellular
environment in which said nucleic acid or protein may be found in
nature. The term encompasses both nucleic acids originally obtained
from a different organism or cell type than the cell type in which
it is expressed, and also nucleic acids that are obtained from the
same cell line as the cell line in which it is expressed.
[0030] A prokaryotic cell has been "transformed" by an exogenous
nucleic acid when such exogenous nucleic acid has been introduced
inside the cell membrane. Exogenous DNA may or may not be
integrated (covalently linked) into chromosomal DNA making up the
genome of the cell. The exogenous DNA may be maintained on an
episomal element, such as a plasmid.
[0031] An "expression cassette" is a nucleic acid construct,
generated recombinantly or synthetically, with nucleic acid
elements that are capable of effecting expression of a structural
gene in hosts compatible with such sequences. Expression cassettes
include at least promoters and optionally, transcription
termination signals. Typically, the recombinant expression cassette
includes a nucleic acid to be transcribed (e.g., a nucleic acid
encoding a desired polypeptide), and a promoter. Additional factors
necessary or helpful in effecting expression may also be used as
described herein. For example, an expression cassette can also
include nucleotide sequences that encode a signal sequence that
directs secretion of an expressed protein from the host cell.
Transcription termination signals, enhancers, and other nucleic
acid sequences that influence gene expression, can also be included
in an expression cassette.
[0032] The term "over expression" refers to the situation when one
or more components of cell may be present at a higher than normal
cellular level (i.e., higher than the concentration known to
usually be present in the cell type exhibiting the protein complex
of interest). For example, the gene encoding a protein may begin to
be overexpressed, or may be amplified (i.e., its gene copy number
may be increased) in certain cells, leading to an increased number
of component molecules within these cells. Typically overexpression
will result in from about 10% to about 15% over the basal
expression level.
[0033] The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two or
more subsequences that are not found in the same relationship to
each other in nature. For instance, the nucleic acid is typically
recombinantly produced, having two or more sequences from unrelated
genes arranged to make a new functional nucleic acid, e.g., a
promoter from one source and a coding region from another source.
Similarly, a heterologous protein indicates that the protein
comprises two or more subsequences that are not found in the same
relationship to each other in nature (e.g., a fusion protein).
[0034] A "promoter" is defined as an array of nucleic acid control
sequences that direct transcription of a nucleic acid. As used
herein, a promoter includes necessary nucleic acid sequences near
the start site of transcription, such as, in the case of a
polymerase II type promoter, a TATA element. A promoter also
optionally includes distal enhancer or repressor elements, which
can be located as much as several thousand base pairs from the
start site of transcription. A "constitutive" promoter is a
promoter that is active under environmental or developmental
regulation. The term "operably linked" refers to a functional
linkage between a nucleic acid expression control sequence (such as
a promoter, or array of transcription factor binding sites) and a
second nucleic acid sequence, wherein the expression control
sequence directs transcription of the nucleic acid corresponding to
the second sequence.
[0035] The term "recombinant" when used with reference to a cell,
or nucleic acid, or vector, indicates that the cell, or nucleic
acid, or vector, has been modified by the introduction of a
heterologous nucleic acid or the alteration of a native nucleic
acid, or that the cell is derived from a cell so modified. Thus,
for example, recombinant cells express genes that are not found
within the native (non-recombinant) form of the cell or express
native genes that are otherwise abnormally expressed, under
expressed or not expressed at all.
[0036] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same, when compared and aligned for maximum correspondence over
a comparison window, as measured using one of the following
sequence comparison algorithms or by manual alignment and visual
inspection. This definition also refers to the complement of a test
sequence, which has a designated percent sequence or subsequence
complementarity when the test sequence has a designated or
substantial identity to a reference sequence. For example, a
designated amino acid percent identity of 86% refers to sequences
or subsequences that have at least about 86% amino acid identity
when aligned for maximum correspondence over a comparison window as
measured using one of the following sequence comparison algorithms
or by manual alignment and visual inspection. Preferably, the
percent identity exists over a region of the sequence that is at
least about 25 amino acids in length, more preferably over a region
that is 50 amino acids in length.
[0037] When percentage of sequence identity is used in reference to
proteins or peptides, it is recognized that residue positions that
are not identical often differ by conservative amino acid
substitutions, where amino acids residues are substituted for other
amino acid residues with similar chemical properties (e.g., charge
or hydrophobicity) and therefore do not change the functional
properties of the molecule. Where sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Means for making this adjustment are well known to those of skill
in the art. Typically this involves scoring a conservative
substitution as a partial rather than a full mismatch, thereby
increasing the percentage sequence identity. Thus, for example,
where an identical amino acid is given a score of 1 and a
non-conservative substitution is given a score of zero, a
conservative substitution is given a score between zero and 1. The
scoring of conservative substitutions is calculated according to,
e.g., the algorithm of Meyers & Miller, Computer Applic. Biol.
Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE
(Intelligenetics, Mountain View, Calif., USA).
[0038] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identity for the
test sequence(s) relative to the reference sequence, based on the
designated or default program parameters.
[0039] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 25 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection (see, e.g., Ausubel et al.,
supra).
[0040] One example of a useful algorithm is PILEUP. PILEUP creates
a multiple sequence alignment from a group of related sequences
using progressive, pairwise alignments to show relationship and
percent sequence identity. It also plots a tree or dendogram
showing the clustering relationships used to create the alignment.
PILEUP uses a simplification of the progressive alignment method of
Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The method
used is similar to the method described by Higgins & Sharp,
CABIOS 5:151-153 (1989). The program can align up to 300 sequences,
each of a maximum length of 5,000 nucleotides or amino acids. The
multiple alignment procedure begins with the pairwise alignment of
the two most similar sequences, producing a cluster of two aligned
sequences. This cluster is then aligned to the next most related
sequence or cluster of aligned sequences. Two clusters of sequences
are aligned by a simple extension of the pairwise alignment of two
individual sequences. The final alignment is achieved by a series
of progressive, pairwise alignments. The program is run by
designating specific sequences and their amino acid or nucleotide
coordinates for regions of sequence comparison and by designating
the program parameters. Using PILEUP, a reference sequence is
compared to other test sequences to determine the percent sequence
identity relationship using the following parameters: default gap
weight (3.00), default gap length weight (0.10), and weighted end
gaps. PILEUP can be obtained from the GCG sequence analysis
software package, e.g., version 7.0 (Devereaux et al., Nuc. Acids
Res. 12:387-395 (1984)).
[0041] Another example of algorithm that is suitable for
determining percent sequence identity (i.e., substantial similarity
or identity) is the BLAST algorithm, which is described in Altschul
et al., J. Mol. Biol. 215:403-410 (1990). Software for performing
BLAST analyses is publicly available through the National Center
for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This
algorithm involves first identifying high scoring sequence pairs
(HSPs) by identifying short words of length W in the query
sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al, supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are then extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues, always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) of 10, M=5, N=4, and a comparison of both strands. For amino
acid sequences, the BLASTP program uses as default parameters a
wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915 (1989)).
[0042] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin &
Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to an ATP binding cassette nucleic acid
sequence, such as MXR1, if the smallest sum probability in a
comparison of the test nucleic acid to the MXR1 nucleic acid is
less than about 0.1, more preferably less than about 0.01, and most
preferably less than about 0.001.
[0043] The term "substantial identity" or "substantial similarity"
in the context of a polypeptide indicates that a polypeptide
comprises a sequence with at least 70% sequence identity to a
reference sequence, or preferably 80%, or more preferably 85%
sequence identity to the reference sequence, or most preferably 90%
identity over a comparison window of about 10-20 amino acid
residues.
[0044] An indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the antibodies raised against the polypeptide encoded by the
second nucleic acid. Thus, a polypeptide is typically substantially
identical to a second polypeptide, for example, where the two
peptides differ only by conservative substitutions. Another
indication that two nucleic acid sequences are substantially
identical is that the two molecules or their complements hybridize
to each other under stringent conditions.
[0045] "Bind(s) substantially" refers to complementary
hybridization between a probe nucleic acid and a target nucleic
acid and embraces minor mismatches that can be accommodated by
reducing the stringency of the hybridization media to achieve the
desired detection of the target polynucleotide sequence.
[0046] The phrase "hybridizing specifically to" refers to the
binding, duplexing, or hybridizing of a molecule only to a
particular nucleotide sequence under stringent conditions when that
sequence is present in a complex mixture (e.g., total cellular) DNA
or RNA. The term "stringent conditions" refers to conditions under
which a probe will hybridize to its target subsequence, but to no
other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. Generally, stringent
conditions are selected to be about 5.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength and pH. The T.sub.m is the temperature
(under defined ionic strength, pH, and nucleic acid concentration)
at which 50% of the probes complementary to the target sequence
hybridize to the target sequence at equilibrium. (As the target
sequences are generally present in excess, at T.sub.m, 50% of the
probes are occupied at equilibrium). Typically, stringent
conditions will be those in which the salt concentration is less
than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium
ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short probes (e.g.,
10 to 50 nucleotides) and at least about 60.degree. C. for long
probes (e.g., greater than 50 nucleotides). Stringent conditions
may also be achieved with the addition of destabilizing agents such
as formamide.
[0047] The phrases "specifically binds to a protein" or
"specifically immunoreactive with," when referring to an antibody
refers to a binding reaction which is determinative of the presence
of the protein in the presence of a heterogeneous population of
proteins and other biologics. Thus, under designated immunoassay
conditions, the specified antibodies bind preferentially to a
particular protein and do not bind in a significant amount to other
proteins present in the sample. Specific binding to a protein under
such conditions requires an antibody that is selected for its
specificity for a particular protein. A variety of immunoassay
formats may be used to select antibodies specifically
immunoreactive with a particular protein. For example, solid-phase
ELISA immunoassays are routinely used to select monoclonal
antibodies specifically immunoreactive with a protein. See Harlow
and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor
Publications, New York, for a description of immunoassay formats
and conditions that can be used to determine specific
immunoreactivity. For determination of specific binding of an
anti-ABC antibody, an immunoblot assay is preferred.
[0048] A "conservative substitution," when describing a protein
refers to a change in the amino acid composition of the protein
that does not substantially alter the protein's activity. Thus,
"conservatively modified variations" of a particular amino acid
sequence refers to amino acid substitutions of those amino acids
that are not critical for protein activity or substitution of amino
acids with other amino acids having similar properties (e.g.,
acidic, basic, positively or negatively charged, polar or
non-polar, etc.) such that the substitutions of even critical amino
acids do not substantially alter activity. Conservative
substitution tables providing functionally similar amino acids are
well known in the art. The following six groups each contain amino
acids that are conservative substitutions for one another:
[0049] 1) Alanine (A), Serine (S), Threonine (T);
[0050] 2) Aspartic acid (D), Glutamic acid (E);
[0051] 3) Asparagine (N), Glutamine (Q);
[0052] 4) Arginine (R), Lysine (K);
[0053] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
and
[0054] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0055] See also, Creighton (1984) Proteins, W.H. Freeman and
Company. One of skill in the art will appreciate that the
above-identified substitutions are not the only possible
conservative substitutions. For example, one may regard all charged
amino acids as conservative substitutions for each other whether
they are positive or negative. In addition, individual
substitutions, deletions or additions which alter, add or delete a
single amino acid or a small percentage of amino acids in an
encoded sequence are also "conservatively modified variations".
[0056] The terms "isolated," "purified," or "biologically pure"
refer to material that is substantially or essentially free from
components that normally accompany it as found in the native
state.
[0057] An "expression vector" is a nucleic acid construct,
generated recombinantly or synthetically, with a series of
specified nucleic acid elements that permit transcription of a
particular nucleic acid in a host cell. The expression vector can
be part of a plasmid, virus, or nucleic acid fragment. Typically,
the expression vector includes a nucleic acid to be transcribed or
operably linked to a promoter.
[0058] The phrase "a nucleic acid sequence encoding" refers to a
nucleic acid which contains sequence information for a structural
RNA such as rRNA, a tRNA, or the primary amino acid sequence of a
specific protein or peptide, or a binding site for a transacting
regulatory agent. This phrase specifically encompasses degenerate
codons (i.e., different codons which encode a single amino acid) of
the native sequence or sequences which may be introduced to conform
with codon preference in a specific host cell.
[0059] "Antisense sequence or antisense nucleic acids" are used
interchangeably and refer to sequences of nucleic acids that are
complementary to the coding mRNA nucleic acid sequence. The phrase
specifically encompasses nucleic acid sequences that bind to mRNA
or portions thereof to block transcription of mRNA by
ribosomes.
[0060] "Conferring mitoxantrone resistance" refers to the situation
in which the expression of a protein in a cell makes the cell
resistant to a particular drug or antibiotic. For example, cells
where an ABC gene is overexpressed or amplified in certain breast
and colon cancer cell lines will be resistant to the
chemotherapeutic drug mitoxantrone, and to a lesser extent
daunorubicin, i.e., the drugs will have no effect on the cell.
[0061] "Genetically altered" refers to a protein, cell, nucleic
acid or other biological molecule that has been recombinantly or
otherwise manipulated such that it is no longer in its native
state.
[0062] The phrase "basal expression levels" refers to the normal,
base or fundamental level of protein expression in a cell in its
usual environment.
[0063] "Enhancing element" refers to a component in a cell that
enhances or increases the basal or normal protein expression level.
Such an element will cause a cell to express more of a protein than
it would under natural or normal conditions.
[0064] "Growth inhibition" refers to cell death or a slowing down
or suppression of cell growth, biological function, or division.
The growth inhibition can be caused by chemical or physical
means.
[0065] Genes Encoding ATP-Binding Cassette Protein
[0066] A. General Recombinant DNA Methods
[0067] This invention relies on routine techniques in the field of
recombinant genetics to produce the MXR1 nucleic acids of the
present invention. Basic texts disclosing the general methods of
use in this invention include Sambrook et al., Molecular Cloning, A
Laboratory Manual (2.sup.nd ed. 1989); Kriegler, Gene Transfer and
Expression: A Laboratory Manual (1990); and Current Protocols in
Molecular Biology (Ausubel et al., eds., 1994).
[0068] For nucleic acids, sizes are given in either kilobases (kb)
or base pairs (bp). These are estimates derived from agarose or
acrylamide gel electrophoresis, from sequenced nucleic acids, or
from published DNA sequences. For proteins, sizes are given in
kilodaltons (kDa) or amino acid residue numbers. Protein sizes are
estimated from gel electrophoresis, from sequenced proteins, from
derived amino acid sequences, or from published protein
sequences.
[0069] Oligonucleotides that are not commercially available can be
chemically synthesized according to the solid phase phosporamidite
triester method first described by Beaucage & Caruthers,
Tetrahedron Letts. 22:1859-1862 (1981), using an automated
synthesizer, as described in Van Devanter et al., Nucleic Acids
Res. 12:6159-6168 (1984). Purification of oligonucleotides is by
either native acrylamide gel electrophoresis or by anion exchange
HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149
(1983).
[0070] The sequence of the cloned genes and synthetic
oligonucleotides can be verified after cloning using, e.g., the
chain termination method for sequencing double-stranded templates
of Wallace et al., Gene 16:21-26 (1981).
[0071] B. Cloning Methods for the Isolation of Nucleotide Sequences
Encoding MXR1
[0072] In general, the nucleic acid sequences encoding the ATP
binding cassette proteins and related nucleic acid homologs are
cloned from cDNA and genomic DNA libraries by hybridization with a
probe, or isolated using amplification techniques with
oligonucleotide primers. For example, ABC proteins are typically
isolated from mammalian DNA libraries by hybridizing with a nucleic
acid probe, the sequence of which can be derived from SEQ ID NO: 1
or 3. A suitable tissue from which ABC proteins and cDNA can be
isolated is fetal brain or liver, and preferably placenta.
[0073] Amplification techniques using primers can also be used to
amplify and isolate ABC nucleic acids from DNA or RNA. The
degenerate primers encoding the following amino acid sequences can
also be use to amplify a sequence of MXR1. SEQ ID NOS: 5 & 6
(Dieffanfach & Dveksler, PCR Primer: A Laboratory Manual
(1995)). These primers can be used, e.g., to amplify either the
full length sequence or a probe of one to a hundred nucleotides,
which is then used to screen a mammalian library for full length
MXR1.
[0074] Nucleic acids encoding ABC proteins can also be isolated
from expression libraries using antibodies as probes. Such
polyclonal or monoclonal antibodies can be raised using the
sequence of SEQ ID NO: 2 or 4
[0075] MXR polymorphic variants, alleles, and interspecies homologs
that are substantially identical to MXR1 and MXR2 can also be
isolated using MXR1 and MXR2 nucleic acid probes, and
oligonucleotides under stringent hybridization conditions, by
screening libraries. Alternatively, expression libraries can be
used to clone MXR1 and MXR2 polymorphic variants, alleles, and
interspecies homologs, by detecting homologs immunologically with
antisera or purified antibodies made against MXR1 or MXR2, which
also recognize and selectively bind to the MXR1 or MXR2
homolog.
[0076] To make a cDNA library, one should choose a source that is
rich in the MXR mRNA, e.g., human colon carcinoma cells. Placenta
tissue or fetal brain or liver tissue. The mRNA is then made into
cDNA using reverse transcriptase, ligated into a recombinant
vector, and transfected into a recombinant host for propagation,
screening, and cloning. Methods for making and screening cDNA
libraries are well known (see, e.g., Gubler & Hoffman, Gene
25:263-269 (1983); Sambrook et al., supra; Ausubel et al.,
supra).
[0077] For a genetic library, the DNA is extracted from the tissue
and either mechanically sheared or enzymatically digested to yield
fragments of about 12-20 kb. The fragments are then separated by
gradient centrifugation from undesired sizes and are constructed in
bacteriophage lambda vectors. These vectors and phage are described
in Benton & Davis, Science 196:180-182 (1977). Colony
hybridization is carried out as generally described in Grunstein et
al., PNAS USA, 72:3961-3965 (1975).
[0078] An alternative method of isolating MXR nucleic acids and
their homologs combines the use of synthetic oligonucleotide
primers and amplification of an RNA or DNA template (see U.S. Pat.
Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and
Applications (Innis et al., eds. 1990)). Methods such as polymerase
chain reaction (PCR) and ligase chain reaction (LCR) can be used to
amplify nucleic acid sequences of ABC proteins directly from mRNA,
from cDNA, from genomic libraries or cDNA libraries. Degenerate
oligonucleotides can be designed to amplify ABC homologs using the
sequences provided herein. Restriction endonuclease sites can be
incorporated into the primers. PCR or other in vitro amplification
methods may be useful, for example, to clone nucleic acid sequences
that code for proteins to be expressed, to make nucleic acids to
use as probes for detecting the presence of ABC protein encoding
mRNA in physiological samples, for nucleic acid sequencing, or for
other purposes. Genes amplified by the PCR reaction can be purified
from agarose gels and cloned into an appropriate vector.
[0079] Gene expression of ABC proteins can also be analyzed by
techniques known in the art, e.g., reverse transcription and
amplification of mRNA, isolation of total RNA or polyA.sup.+ RNA,
northern blotting, dot blotting, in situ hybridization, RNAse
protection, probing DNA microchip arrays, and the like.
[0080] Synthetic oligonucleotides can be used to construct
recombinant ABC genes for use as probes or for expression of
protein. This method is performed using a series of overlapping
oligonucleotides usually 40-120 bp in length, representing both the
sense and nonsense strands of the gene. These DNA fragments are
then annealed, ligated and cloned. Alternatively, amplification
techniques can be used with precise primers to amplify a specific
subsequence of the ABC nucleic acid. The specific subsequence is
then ligated into an expression vector.
[0081] The nucleic acid encoding the MXR1 protein is typically
cloned into intermediate vectors before transformation into
prokaryotic or eukaryotic cells for replication and/or expression.
These intermediate vectors are typically prokaryote vectors, e.g.,
plasmids or shuttle vectors.
[0082] Expression of ATP-Binding Cassette Proteins
[0083] To obtain high level expression of a cloned gene or nucleic
acid, such as those cDNAs encoding the ABC protein MXR1, one
typically subclones MXR into an expression vector that contains a
strong promoter to direct transcription, a
transcription/translation terminator, and if for a nucleic acid
encoding a protein, a ribosome binding site for translational
initiation. Suitable bacterial promoters are well known in the art
and described, e.g., in Sambrook et al. and Ausubel et al.
Bacterial expression systems for expressing MXR1 are available in,
e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene
22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983)). Kits
for such expression systems are commercially available. Eukaryotic
expression systems for mammalian cells, yeast, and insect cells are
well known in the art and are also commercially available.
[0084] The promoter used to direct expression of a heterologous
nucleic acid depends on the particular application. The promoter is
preferably positioned about the same distance from the heterologous
transcription start site as it is from the transcription start site
in its natural setting. As is known in the art, however, some
variation in this distance can be accommodated without loss of
promoter function.
[0085] In addition to the promoter, the expression vector typically
contains a transcription unit or expression cassette that contains
all the additional elements required for the expression of the MXR1
encoding nucleic acid in host cells. A typical expression cassette
thus contains a promoter operably linked to the nucleic acid
sequence encoding an ABC protein and signals required for efficient
polyadenylation of the transcript, ribosome binding sites, and
translation termination. The nucleic acid sequence encoding MXR1
may typically be linked to a cleavable signal peptide sequence to
promote secretion of the encoded protein by the transformed cell.
Such signal peptides would include, among others, the signal
peptides from tissue plasminogen activator, insulin, and neuron
growth factor, and juvenile hormone esterase of Heliothis
virescens. Additional elements of the cassette may include
enhancers and, if genomic DNA is used as the structural gene,
introns with functional splice donor and acceptor sites.
[0086] In addition to a promoter sequence, the expression cassette
should also contain a transcription termination region downstream
of the structural gene to provide for efficient termination. The
termination region may be obtained from the same gene as the
promoter sequence or may be obtained from different genes.
[0087] The particular expression vector used to transport the
genetic information into the cell is not particularly critical. Any
of the conventional vectors used for expression in eukaryotic or
prokaryotic cells may be used. Standard bacterial expression
vectors include plasmids such as pBR322 based plasmids, pSKF,
pET23D, and fusion expression systems such as GST and LacZ. Epitope
tags can also be added to recombinant proteins to provide
convenient methods of isolation, e.g., c-myc.
[0088] Expression vectors containing regulatory elements from
eukaryotic viruses are typically used in eukaryotic expression
vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors
derived from Epstein-Barr virus. Other exemplary eukaryotic vectors
include pMSG, pAV009/A.sup.+, pMTO10/A.sup.+, pMAMneo-5,
baculovirus pDSVE, and any other vector allowing expression of
proteins under the direction of the SV40 early promoter, SV40 later
promoter, metallothionein promoter, murine mammary tumor virus
promoter, Rous sarcoma virus promoter, polyhedrin promoter, or
other promoters shown effective for expression in eukaryotic
cells.
[0089] Some expression systems have markers that provide gene
amplification such as thymidine kinase, hygromycin B
phosphotransferase, and dihydrofolate reductase. Alternatively,
high yield expression systems not involving gene amplification are
also suitable, such as using a baculovirus vector in insect cells,
with an MXR1 encoding sequence under the direction of the
polyhedrin promoter or other strong baculovirus promoters.
[0090] The elements that are typically included in expression
vectors also include a replicon that functions in E. coli, a gene
encoding antibiotic resistance to permit selection of bacteria that
harbor recombinant plasmids, and unique restriction sites in
nonessential regions of the plasmid to allow insertion of
eukaryotic sequences. The particular antibiotic resistance gene
chosen is not critical, any of the many resistance genes known in
the art are suitable. The prokaryotic sequences are preferably
chosen such that they do not interfere with the replication of the
DNA in eukaryotic cells, if necessary.
[0091] Standard transfection methods are used to produce bacterial,
mammalian, yeast or insect cell lines that express large quantities
of MXR protein, which are then purified using standard techniques
(see, e.g., Colley et al., J. Biol. Chem. 264:17619-17622 (1989);
Guide to Protein Purification, in Methods in Enzymology, vol. 182
(Deutscher, ed., 1990)). Transformation of eukaryotic and
prokaryotic cells are performed according to standard techniques
(see, e.g., Morrison, J. Bact. 132:349-351 (1977); Clark-Curtiss
& Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds,
1983).
[0092] Any of the well known procedures for introducing foreign
nucleotide sequences into host cells may be used. These include the
use of calcium phosphate transfection, polybrene, protoplast
fusion, electroporation, liposomes, microinjection, plasma vectors,
viral vectors and any of the other well known methods for
introducing cloned genomic DNA, cDNA, synthetic DNA or other
foreign genetic material into a host cell (see, e.g., Sambrook et
al., supra). It is only necessary that the particular genetic
engineering procedure used be capable of successfully introducing
at least one gene into the host cell capable of expressing MXR
protein.
[0093] Another mode of expression for ABC proteins involves
transactivation, which describes a method of activating (i.e.,
turning on) and amplifying an endogenous gene encoding a desired
product, such as MXR1, in a transfected cell (See, e.g., U.S. Pat.
No. 5,733,761).
[0094] DNA sequences that are not normally functionally linked to
the endogenous gene, can be introduced by homologous recombination
with genomic DNA. The DNA sequences would be inserted into the host
genome at or near the endogenous gene and serve to alter (e.g.,
activate) the expression of the endogenous gene and further allow
selection of cells in which the activated endogenous gene is
amplified.
[0095] The transactivation can be used to target different events
in the cell by a simple insertion of a regulatory sequence that
places the endogenous gene under the control of the new regulatory
sequence (for example, by insertion of either a promoter or an
enhancer, both upstream of an endogenous gene). Additionally the
transactivation protocols can be used to delete a regulatory
element or replace an existing element. For example, a tissue
specific enhancer can be replaced by an enhancer that has broader
or different cell-type specificity. In all cases, the targeting
event can be identified by the use of one or more selectable
markers that are physically associated with the targeting DNA
sequence, allowing for selection of cells in which the exogenous
DNA sequence has been integrated into the host cell genome. (see,
e.g., U.S. Pat. No. 5,733,761.)
[0096] In another embodiment, the invention includes polymorphic
alleles of MXR1. In addition, those of skill can readily create
muteins or analogs of MXR1 based on comparisons with the mouse
sequence of SEQ ID NO: 4 and conservative amino acid substitutions.
When compared to SEQ ID NO: 2, a protein that exhibits conservative
substitutions, as described above, is a protein of the invention.
Such substitutions will alter the sequence of the protein from that
provided in SEQ ID NO:2, but will not markedly change the
biological activity of the molecule. For example, the serine at
position 519 may be changed to a threonine; the alanine at position
529 may be changed to a threonine; the isoleucine at position 550
may be changed to leucine; and the alanine at position 597 may be
changed to valine. These substitutions are provided by way of
illustration and for clarity of understanding and not by way of
limitation. It will be readily apparent to those of ordinary skill
in the art, in light of the teachings of the invention, that
certain changes may be made to the MXR1 protein sequence without
changing its biological activity.
[0097] After the expression vector is introduced into the cells,
the transfected cells are cultured under conditions favoring
expression of MXR1 proteins, which are recovered from the culture
using standard techniques identified below.
[0098] Purification of ATP-Binding Cassette Proteins
[0099] Once expressed the MXR1 proteins can be purified. Either
naturally occurring or recombinant MXR1 protein can be purified for
use in functional assays. Preferably, recombinant MXR1 is purified.
Naturally occurring MXR1 protein is purified, e.g., from mammalian
tissue such as placenta, fetal brain or liver tissues and any other
source of an MXR1 homolog. Recombinant MXR1 is purified from any
suitable expression system.
[0100] MXR1 may be purified to substantial purity by standard
techniques, including selective precipitation with such substances
as ammonium sulfate; column chromatography, immunopurification
methods, and others (see, e.g., Scopes, Protein Purification:
Principles and Practice (1982); U.S. Pat. No. 4,673,641; Ausubel et
al., supra; and Sambrook et al., supra).
[0101] A number of procedures can be employed when recombinant MXR1
is being purified. For example, proteins having established
molecular adhesion properties can be reversibly fused to MXR1. With
the appropriate ligand, MXR1 can be selectively adsorbed to a
purification column and then freed from the column in a relatively
pure form. The fused protein is then removed by enzymatic activity.
Finally, MXR1 protein could be purified using immunoaffinity
columns.
[0102] A. Purification of MXR1 from Recombinant Bacteria
[0103] Recombinant proteins are expressed by transformed bacteria
in large amounts, typically after promoter induction; but
expression can be constitutive. Promoter induction with IPTG is a
one example of an inducible promoter system. Bacteria are grown
according to standard procedures in the art. Fresh or frozen
bacteria cells are used for isolation of protein.
[0104] Proteins expressed in bacteria may form insoluble aggregates
("inclusion bodies"). Several protocols are suitable for
purification of MXR1 inclusion bodies. For example, purification of
inclusion bodies typically involves the extraction, separation
and/or purification of inclusion bodies by disruption of bacterial
cells, e.g., by incubation in a buffer of 50 mM TRIS/HCL pH 7.5, 50
mM NaCl, 5 mM MgCl.sub.2, 1 mM DTT, 0.1 mM ATP, and 1 mM PMSF. The
cell suspension can be lysed using 2-3 passages through a French
Press, homogenized using a Polytron (Brinkman Instruments) or
sonicated on ice. Alternate methods of lysing bacteria are apparent
to those of skill in the art (see, e.g., Sambrook et al., supra;
Ausubel et al., supra).
[0105] If necessary, the inclusion bodies are solubilized, and the
lysed cell suspension is typically centrifuged to remove unwanted
insoluble matter. Proteins that formed the inclusion bodies may be
renatured by dilution or dialysis with a compatible buffer.
Suitable solvents include, but are not limited to urea (from about
4 M to about 8 M), formamide (at least about 80%, volume/volume
basis), and guanidine hydrochloride (from about 4 M to about 8 M).
Some solvents which are capable of solubilizing aggregate-forming
proteins, for example SDS (sodium dodecyl sulfate), 70% formic
acid, are inappropriate for use in this procedure due to the
possibility of irreversible denaturation of the proteins,
accompanied by a lack of immunogenicity and/or activity. Although
guanidine hydrochloride and similar agents are denaturants, this
denaturation is not irreversible and renaturation may occur upon
removal (by dialysis, for example) or dilution of the denaturant,
allowing re-formation of immunologically and/or biologically active
protein. Other suitable buffers are known to those skilled in the
art. MXR1 is separated from other bacterial proteins by standard
separation techniques, e.g., with Ni-NTA agarose resin.
[0106] Alternatively, it is possible to purify MXR1 protein from
bacteria periplasm. After lysis of the bacteria, when MXR1 is
exported into the periplasm of the bacteria, the periplasmic
fraction of the bacteria can be isolated by cold osmotic shock in
addition to other methods known to those of skill in the art. To
isolate recombinant proteins from the periplasm, the bacterial
cells are centrifuged to form a pellet. The pellet is resuspended
in a buffer containing 20% sucrose. To lyse the cells, the bacteria
are centrifuged and the pellet is resuspended in ice-cold 5 mM
MgSO.sub.4 and kept in an ice bath for approximately 10 minutes.
The cell suspension is centrifuged and the supernatant decanted and
saved. The recombinant proteins present in the supernatant can be
separated from the host proteins by standard separation techniques
well known to those of skill in the art.
[0107] B. Standard Protein Separation Techniques for Purifying MXR1
Solubility Fractionation
[0108] Often as an initial step, particularly if the protein
mixture is complex, an initial salt fractionation can separate many
of the unwanted host cell proteins (or proteins derived from the
cell culture media) from the recombinant protein of interest. The
preferred salt is ammonium sulfate. Ammonium sulfate precipitates
proteins by effectively reducing the amount of water in the protein
mixture. Proteins then precipitate on the basis of their
solubility. The more hydrophobic a protein is, the more likely it
is to precipitate at lower ammonium sulfate concentrations. A
typical protocol includes adding saturated ammonium sulfate to a
protein solution so that the resultant ammonium sulfate
concentration is between 20-30%. This concentration will
precipitate the most hydrophobic of proteins. The precipitate is
then discarded (unless the protein of interest is hydrophobic) and
ammonium sulfate is added to the supernatant to a concentration
known to precipitate the protein of interest. The precipitate is
then solubilized in buffer and the excess salt removed if
necessary, either through dialysis or diafiltration. Other methods
that rely on solubility of proteins, such as cold ethanol
precipitation, are well known to those of skill in the art and can
be used to fractionate complex protein mixtures.
[0109] Size Differential Filtration
[0110] The molecular weight of MXR1 can be used to isolated it from
proteins of greater and lesser size using ultrafiltration through
membranes of different pore size (for example, Amicon or Millipore
membranes). As a first step, the protein mixture is ultrafiltered
through a membrane with a pore size that has a lower molecular
weight cut-off than the molecular weight of the protein of
interest. The retentate of the ultrafiltration is then
ultrafiltered against a membrane with a molecular cut off greater
than the molecular weight of the protein of interest. The
recombinant protein will pass through the membrane into the
filtrate. The filtrate can then be chromatographed as described
below.
[0111] Column Chromatography
[0112] MXR1 can also be separated from other proteins on the basis
of its size, net surface charge, hydrophobicity, and affinity for
ligands. In addition, antibodies raised against proteins can be
conjugated to column matrices and the proteins immunopurified. All
of these methods are well known in the art. It will be apparent to
one of skill that chromatographic techniques can be performed at
any scale and using equipment from many different manufacturers
(e.g., Pharmacia Biotech).
[0113] Antisense Applications for ATP-Binding Cassette Proteins
[0114] Gene regulation in MXR1 can be downregulated or entirely
inhibited by the use of antisense molecules. An "antisense sequence
or antisense nucleic acid" is a nucleic acid that is complementary
to the coding MXR1 mRNA nucleic acid sequence or a subsequence
thereof. Binding of the antisense molecule to the MXR1 mRNA
interferes with normal translation of MXR1. The antisense molecule
can be an endogenous or an exogenous complement to an mRNA. It can
also be ribozyme or a ribozyme combined with a mRNA complement.
[0115] In conventional antisense technology, a nucleic acid segment
from the desired gene is cloned and operably linked to a promoter
sequence such that the anti-sense strand of RNA will be
transcribed. The construct is then transformed into cells and the
anti-sense strand of RNA is produced. (see, e.g., Sheehy et al.,
Proc. Nat. Acad. Sci. USA 85:8805-8809 (1988), and Hiatt, et al.,
U.S. Pat. No. 4,801,340.)
[0116] The nucleic acid segment to be introduced in antisense
suppression generally will be substantially identical to at least a
portion of the endogenous gene or gene to be repressed, but need
not be identical. The vectors can thus be designed such that the
inhibitory effect applies to other proteins within a family of
genes exhibiting homology or substantial homology to the target
gene. The introduced sequence also need not be full length relative
to either the primary transcription product or fully processed
mRNA. Generally, higher homology can be used to compensate for the
use of a shorter sequence. Furthermore, the introduced sequence
need not have the same intron or exon pattern, and homology of
non-coding segments will be equally effective.
[0117] Absolute complementarity of the antisense molecule, although
preferred, is not required. A sequence "complementary" to a portion
of an RNA, as referred to herein, means a sequence having
sufficient complementarity to be able to hybridize with the RNA,
forming a stable duplex; in the case of double-stranded antisense
nucleic acids, a single strand of the duplex DNA may thus be
tested, or triplex formation may be assayed. The ability to
hybridize will depend on both the degree of complementarity and the
length of the antisense nucleic acid. Generally, the longer the
hybridizing nucleic acid, the more base mismatches with an RNA it
may contain and still form a stable duplex (or triplex, as the case
may be). One skilled in the art can ascertain a tolerable degree of
mismatch by use of standard procedures to determine the melting
point of the hybridized complex. Antisense oligonucleotides
complementary to mRNA coding regions are less efficient inhibitors
of translation than oligonucleotides that are complementary to 5'-
or 3'-untranslated sequence, but should be used in accordance with
the instant invention. The antisense nucleic acids should be at
least six nucleotides in length, and are preferably
oligonucleotides ranging from 6 to about 50 nucleotides in length.
In specific aspects, the oligonucleotide is at least 10
nucleotides, preferably at least 17 nucleotides, more preferably at
least 25 nucleotides or most preferably at least 50
nucleotides.
[0118] Thus, in accordance with preferred embodiments of this
invention, preferred antisense molecules include oligonucleotides
and oligonucleotide analogs that are hybridizable with MXR1 mRNA.
This relationship is commonly denominated as "antisense." The
oligonucleotides and oligonucleotide analogs are able to inhibit
the function of the RNA, either its translation into protein, its
translocation into the cytoplasm, or any other activity necessary
to its overall biological function. The failure of the messenger
RNA to perform all or part of its function results in a reduction
or complete inhibition of expression of MXR polypeptides.
[0119] The mechanisms above also work with exogenous antisense
molecules that are modified to be nuclease resistant. Therefore, in
the context of this invention, the term "oligonucleotide" refers to
a polynucleotide formed from naturally-occurring bases and/or
cyclofuranosyl groups joined by native phosphodiester bonds. This
term effectively refers to naturally-occurring species or synthetic
species formed from naturally-occurring subunits or their close
homologs. The term "oligonucleotide" may also refer to moieties
which function similarly to oligonucleotides, but which have non
naturally-occurring portions. Thus, oligonucleotides may have
altered sugar moieties or inter-sugar linkages. Exemplary among
these are the phosphorothioate and other sulfur containing species
which are known for use in the art. In accordance with some
preferred embodiments, at least one of the phosphodiester bonds of
the oligonucleotide has been substituted with a structure which
functions to enhance the ability of the compositions to penetrate
into the region of cells where the RNA whose activity is to be
modulated is located. It is preferred that such substitutions
comprise phosphorothioate bonds, methyl phosphonate bonds, or short
chain alkyl or cycloalkyl structures. In accordance with other
preferred embodiments, the phosphodiester bonds are substituted
with structures which are, at once, substantially non-ionic and
non-chiral, or with structures which are chiral and
enantiomerically specific. Persons of ordinary skill in the art
will be able to select other linkages for use in the practice of
the invention.
[0120] Oligonucleotides may also include species which include at
least some modified base forms. Thus, purines and pyrimidines other
than those normally found in nature may be so employed. Similarly,
modifications on the furanosyl portions of the nucleotide subunits
may also be effected, as long as the essential tenets of this
invention are adhered to. Examples of such modifications are
2'-O-alkyl- and 2'-halogen-substituted nucleotides. Some specific
examples of modifications at the 2' position of sugar moieties
which are useful in the present invention are OH, SH, SCH.sub.3, F,
OCH.sub.3, OCN, O(CH.sub.2)[n]NH.sub.2 or O(CH.sub.2)[n]CH.sub.3,
where n is from 1 to about 10, and other substituents having
similar properties.
[0121] Such oligonucleotides are best described as being
functionally interchangeable with natural oligonucleotides or
synthesized oligonucleotides along natural lines, but which have
one or more differences from natural structure. All such analogs
are comprehended by this invention so long as they function
effectively to hybridize with messenger RNA of MXR1 to inhibit the
function of that RNA.
[0122] The oligonucleotides in accordance with this invention
preferably comprise from about 3 to about 50 subunits. It is more
preferred that such oligonucleotides and analogs comprise from
about 8 to about 25 subunits and still more preferred to have from
about 12 to about 20 subunits. As will be appreciated, a subunit is
a base and sugar combination suitably bound to adjacent subunits
through phosphodiester or other bonds. The oligonucleotides used in
accordance with this invention may be conveniently and routinely
made through the well-known technique of solid phase synthesis.
Equipment for such synthesis is sold by several vendors, including
Applied Biosystems. Any other means for such synthesis may also be
employed, however, the actual synthesis of the oligonucleotides is
well within the talents of the routineer. It is also well known to
prepare other oligonucleotide such as phosphorothioates and
alkylated derivatives.
[0123] Catalytic RNA molecules or ribozymes can be used as a means
to inhibit expression of endogenous genes. It is possible to design
ribozymes that specifically pair with virtually any target RNA and
cleave the phosphodiester backbone at a specific location, thereby
functionally inactivating the target RNA. In carrying out this
cleavage, the ribozyme is not itself altered, and is thus capable
of recycling and cleaving other molecules, making it a true enzyme.
The inclusion of ribozyme sequences within antisense RNAs confers
RNA-cleaving activity upon them, thereby increasing the activity of
the constructs. Ribozymes include but are not limited to any of the
various types, such as hairpin or hammerhead ribozymes. The design
and use of target RNA-specific ribozymes is described in Haseloff
et al., Nature, 334:585-591 (1988).
[0124] Antibodies Binding ATP-Binding Cassette Proteins
[0125] Methods of producing polyclonal and monoclonal antibodies
that react specifically with MXR1 are known to those of skill in
the art. See, e.g., Coligan (1991), CURRENT PROTOCOLS IN
IMMUNOLOGY, Wiley/Greene, NY; and Harlow and Lane; Stites et al.
(eds.) BASIC AND CLINICAL IMMUNOLOGY (4th ed.) Lange Medical
Publications, Los Altos, Calif., and references cited therein;
Goding (1986), MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d
ed.) Academic Press, New York, N.Y.; and Kohler and Milstein
(1975), Nature, 256:495-497. Such techniques include antibody
preparation by selection of antibodies from libraries of
recombinant antibodies in phage or similar vectors. See, Huse et
al. (1989), Science, 246:1275-1281; and Ward et al. (1989), Nature,
341:544-546. For example, in order to produce antisera for use in
an immunoassay, the ATP binding cassette polypeptide partially
encoded by SEQ ID NO: 1 or 3 or a fragment thereof, is isolated as
described herein. For example, recombinant protein is produced in a
transformed cell line. An inbred strain of mice or rabbits is
immunized with the protein using a standard adjuvant, such as
Freund's adjuvant, and a standard immunization protocol.
Alternatively, a synthetic peptide derived from the sequences
disclosed herein and conjugated to a carrier protein can be used an
immunogen. Polyclonal sera are collected and titered against the
immunogen protein in an immunoassay, for example, a solid phase
immunoassay with the immunogen immobilized on a solid support.
Polyclonal antisera with a titer of 10.sup.4 or greater are
selected and tested for their cross reactivity against non-ATP
binding cassette protein, MXR1, or even other homologous proteins
from other organisms, using a competitive binding immunoassay.
Specific monoclonal and polyclonal antibodies and antisera will
usually bind with a K.sub.D of at least about 0.1 mM, more usually
at least about 1 .mu.M, preferably at least about 0.1 .mu.M or
better, and most preferably, 0.01 .mu.M or better.
[0126] A number of MXR1 comprising immunogens may be used to
produce antibodies specifically reactive with MXR1. Recombinant
protein is the preferred immunogen for the production of monoclonal
or polyclonal antibodies. Naturally occurring protein may also be
used either in pure or impure form. Synthetic peptides made using
the protein sequences described herein may also be used as an
immunogen for the production of antibodies to the protein.
Recombinant protein can be expressed in eukaryotic or prokaryotic
cells as described above, and purified as generally described
above. The product is then injected into an animal capable of
producing antibodies. Either monoclonal or polyclonal antibodies
may be generated, for subsequent use in immunoassays to measure the
protein.
[0127] Methods of production of polyclonal antibodies are known to
those of skill in the art. In brief, an immunogen, preferably a
purified protein, is mixed with an adjuvant and animals are
immunized. The animal's immune response to the immunogen
preparation is monitored by taking test bleeds and determining the
titer of reactivity to the ATP binding cassette protein, MXR1. When
appropriately high titers of antibody to the immunogen are
obtained, blood is collected from the animal and antisera are
prepared. Further fractionation of the antisera to enrich for
antibodies reactive to the protein can be done if desired. (See
Harlow and Lane, supra).
[0128] Monoclonal antibodies may be obtained by various techniques
familiar to those skilled in the art. Briefly, spleen cells from an
animal immunized with a desired antigen are immortalized, commonly
by fusion with a myeloma cell (See, Kohler and Milstein, Eur. J.
Immunol. 6:511-519 (1976), incorporated herein by reference).
Alternative methods of immortalization include transformation with
Epstein Barr Virus, oncogenes, or retroviruses, or other methods
well known in the art. Colonies arising from single immortalized
cells are screened for production of antibodies of the desired
specificity and affinity for the antigen, and yield of the
monoclonal antibodies produced by such cells may be enhanced by
various techniques, including injection into the peritoneal cavity
of a vertebrate host. Alternatively, one may isolate DNA sequences
which encode a monoclonal antibody or a binding fragment thereof by
screening a DNA library from human B cells according to the general
protocol outlined by Huse, et al. (1989) Science 246:1275-1281.
[0129] Other suitable techniques involve selection of libraries of
recombinant antibodies in phage or similar vectors (see, e.g., Huse
et al. (1989) Science 246: 1275-1281; and Ward, et al. (1989)
Nature 341: 544-546; and Vaughan et al. (1996) Nature
Biotechnology, 14: 309-314).
[0130] Single chain recombinant versions of antibodies, against
predetermined fragments of ABC polypeptides, such as MXR1, are
raised by immunizing animals, e.g., with conjugates of the
fragments with carrier proteins as described above. Typically, the
immunogen of interest is a peptide of at least about 5 amino acids,
more typically the peptide is 10 amino acids in length, preferably,
the fragment is 15 amino acids in length and more preferably the
fragment is 20 amino acids in length or greater. The peptides are
typically coupled to a carrier protein (e.g., as a fusion protein),
or are recombinantly expressed in an immunization vector. Antigenic
determinants on peptides to which antibodies bind are typically 3
to 10 amino acids in length.
[0131] Once specific antibodies are available, a particular
protein, such as MXR1, can be detected by a variety of immunoassay
methods. For a review of immunological and immunoassay procedures
in general, see Basic and Clinical Immunology 7th Edition (D.
Stites and A. Terr ed.) 1991. Moreover, the immunoassays of the
present invention can be performed in any of several
configurations, which are reviewed extensively in Enzyme
Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Fla. (1980);
"Practice and Theory of Enzyme Immunoassays," Tijssen; and, Harlow
and Lane, each of which is incorporated herein by reference.
[0132] Immunoassays to ATP binding cassette protein MXR1 of the
present invention may use a polyclonal antiserum which was raised
to the protein partially encoded by SEQ ID NO: 1, or a fragment
thereof. This antiserum is selected to have low crossreactivity
against other non-ATP binding cassette proteins and any such
crossreactivity is removed by immunoabsorption prior to use in the
immunoassay.
[0133] In order to produce antisera for use in an immunoassay, the
ATP binding cassette protein of this invention or a fragment
thereof, is isolated as described herein. For example, recombinant
protein is produced in a transformed cell line. An inbred strain of
mice such as Balb/c is immunized with the protein or a peptide
using a standard adjuvant, such as Freund's adjuvant, and a
standard mouse immunization protocol. Alternatively, a synthetic
peptide derived from the sequences disclosed herein and conjugated
to a carrier protein can be used as an immunogen. Polyclonal sera
are collected and titered against the immunogen protein in an
immunoassay, for example, a solid phase immunoassay with the
immunogen immobilized on a solid support. Polyclonal antisera with
a titer of 10.sup.4 or greater are selected and tested for their
cross reactivity against non-MXR1 ABC proteins, such as the human
white gene homolog (see, e.g., Croop, J. M. et al. Gene (1997) 185
(1):77-85) using a competitive binding immunoassay such as the one
described in Harlow and Lane, supra, at pages 570-573 and
below.
Immunological Binding Assays.
[0134] In a preferred embodiment, the ATP binding cassette protein
is detected and/or quantified using any of a number of well
recognized immunological binding assays (see, e.g., U.S. Pat. Nos.
4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of
the general immunoassays, see also Methods in Cell Biology Volume
37: Antibodies in Cell Biology, Asai, ed. Academic Press, Inc., New
York (1993); Basic and Clinical Immunology 7th Edition, Stites
& Terr, eds. (1991). Immunological binding assays (or
immunoassays) typically utilize a "capture agent" to specifically
bind to and often immobilize the analyte (in this case the ATP
binding cassette protein or subsequence). The capture agent is a
moiety that specifically binds to the analyte. In a preferred
embodiment, the capture agent is an antibody that specifically
binds the ATP binding cassette protein. The antibody (anti-ABC
protein MXR1) may be produced by any of a number of means well
known to those of skill in the art and as described above.
[0135] Immunoassays also often utilize a labeling agent to
specifically bind to and label the binding complex formed by the
capture agent and the analyte. The labeling agent may itself be one
of the moieties comprising the antibody/analyte complex. Thus, the
labeling agent may be a labeled ATP binding cassette polypeptide or
a labeled anti-ATP binding cassette antibody. Alternatively, the
labeling agent may be a third moiety, such as another antibody,
that specifically binds to the antibody/ATP binding cassette
complex.
[0136] In a preferred embodiment, the labeling agent is a second
human ATP binding cassette antibody bearing a label. Alternatively,
the second antibody may lack a label, but it may, in turn, be bound
by a labeled third antibody specific to antibodies of the species
from which the second antibody is derived. The second can be
modified with a detectable moiety, such as biotin, to which a third
labeled molecule can specifically bind, such as enzyme-labeled
streptavidin.
[0137] Other proteins capable of specifically binding
immunoglobulin constant regions, such as protein A or protein G may
also be used as the label agent. These proteins are normal
constituents of the cell walls of streptococcal bacteria. They
exhibit a strong non-immunogenic reactivity with immunoglobulin
constant regions from a variety of species (see, generally Kronval,
et al. (1973) J. Immunol., 111: 1401-1406, and Akerstrom, et al.
(1985) J. Immunol., 135: 2589-2542).
[0138] Throughout the assays, incubation and/or washing steps may
be required after each combination of reagents. Incubation steps
can vary from about 5 seconds to several hours, preferably from
about 5 minutes to about 24 hours. However, the incubation time
will depend upon the assay format, analyte, volume of solution,
concentrations, and the like. Usually, the assays will be carried
out at ambient temperature, although they can be conducted over a
range of temperatures, such as 10.degree. C. to 40.degree. C.
Competitive Assay Formats.
[0139] In competitive assays, the amount of analyte (ATP binding
cassette protein) present in the sample is measured indirectly by
measuring the amount of an added (exogenous) analyte (i.e., the ATP
binding cassette protein) displaced (or competed away) from a
capture agent (anti ABC antibody) by the analyte present in the
sample. In one competitive assay, a known amount of, in this case,
the ATP binding cassette protein, MXR1 is added to the sample and
the sample is then contacted with a capture agent, in this case an
antibody that specifically binds to MXR1. The amount of MXR1 bound
to the antibody is inversely proportional to the concentration of
MXR1 present in the sample.
[0140] In a particularly preferred embodiment, the antibody is
immobilized on a solid substrate. The amount of the MXR1 bound to
the antibody may be determined either by measuring the amount of
MXR1 present in an MXR1/antibody complex, or alternatively by
measuring the amount of remaining uncomplexed protein. The amount
of MXR1 protein may be detected by providing a labeled MXR1
molecule.
[0141] A hapten inhibition assay is another preferred competitive
assay. In this assay a known analyte, in this case the ATP binding
cassette protein, MXR1 is immobilized on a solid substrate. A known
amount of anti-MXR1 antibody is added to the sample, and the sample
is then contacted with the immobilized MXR1. In this case, the
amount of anti-MXR1 antibody bound to the immobilized MXR1 is
inversely proportional to the amount of MXR1 present in the sample.
Again the amount of immobilized antibody may be detected by
detecting either the immobilized fraction of antibody or the
fraction of the antibody that remains in solution. Detection may be
direct where the antibody is labeled or indirect by the subsequent
addition of a labeled moiety that specifically binds to the
antibody as described above.
[0142] Immunoassays in the competitive binding format can be used
for crossreactivity determinations. For example, the protein of SEQ
ID NO:2 can be immobilized to a solid support. Proteins are added
to the assay to compete with the binding of the antisera to the
immobilized antigen. The ability of the above proteins to compete
with the binding of the antisera to the immobilized protein is
compared to the protein partially encoded by SEQ ID NO:1 or 3. The
percent crossreactivity for the above proteins is calculated, using
standard calculations. Those antisera with less than 10%
crossreactivity with each of the proteins listed above are selected
and pooled. The cross-reacting antibodies are optionally removed
from the pooled antisera by immunoabsorption with the above-listed
proteins.
[0143] The immunoabsorbed and pooled antisera are then used in a
competitive binding immunoassay as described above to compare a
second protein, such as human white homolog, (GenBank # U34919) to
the immunogen protein (i.e., MXR1 of SEQ ID NO: 2). In order to
make this comparison, the two proteins are each assayed at a wide
range of concentrations and the amount of each protein required to
inhibit 50% of the binding of the antisera to the immobilized
protein is determined. If the amount of protein required is less
than twice the amount of the protein encoded by SEQ ID NO: 2, then
the second protein is said to specifically bind to an antibody
generated to the MXR1 immunogen.
[0144] In addition to using nucleic acid probes for identifying
novel forms of the protein claimed herein, it is possible to use
antibodies to probe expression libraries. This is a well known
technology. (See Young and Davis, 1982 Efficient isolation of genes
using antibody probes Proc. Natl. Acad. Sci., U.S.A. 80:1194-1198.)
In general, a cDNA expression library maybe prepared from
commercially available kits or using readily available components.
Phage vectors are preferred, but a variety of other vectors are
available for the expression of protein. Such vectors include but
are not limited to yeast, animal cells and Xenopus oocytes. One
selects mRNA from a source that is enriched with the target protein
and creates cDNA which is then ligated into a vector and
transformed into the library host cells for immunoscreening.
Screening involves binding and visualization of antibodies bound to
specific proteins on cells or immobilized on a solid support such
as nitrocellulose or nylon membranes. Positive clones are selected
for purification to homogeneity and the isolated cDNA then prepared
for expression in the desired host cells. A good general review of
this technology can be found in Methods of Cell Biology Vol 37
entitled Antibodies in Cell Biology, Ed. D J Asai pp 369-382,
1993.
[0145] Where the antibodies are generated to a short peptide, the
test proteins are optionally denatured to fully test for selective
binding and it may be best to measure the test proteins are against
proteins of similar size, e.g., one would test a full length
monomer against a prototype full length monomer even though the
antisera was generated against a peptide of the prototype monomer.
This simplifies the test and avoids having to take into account
conformational problems and molecular weight/molar concentrations
in the determination of the results from the competitive
immunoassays.
Assays for Detecting ATP-Binding Cassette Protein Activity and for
Identification of Inhibitors of ATP-Binding Cassette Proteins.
[0146] Cells that overexpress ABC proteins (ABCP) have been shown
to be resistant to several chemotherapy drugs. These include
mitoxantrone, several anthracyclines, rhodamine, daunomycin, SN-38
(the active metabolite of CPT-11), topotecan, and bisantrene. To
identify compounds that can reverse the effect of ABCP
overexpression, the concentration of drug that inhibits the
proliferation of resistant cells by 50% (IC50) can be measured by
an assay in the presence and absence of putative inhibitors.
Compounds that cause a significant decrease in the IC50 will
represent inhibitors and be characterized further.
[0147] The assay involves screening for inhibitors of mitoxantrone
resistance in cells that overexpress the MXR1 protein, such as
S1-M1-80 cells, MCF-7 AdVp3000 cells, or MCF-7 MX100 cells (see,
e.g., Lee et al., J. Cell. Biochem. 65(4):513-526 (1997)). The
cells used can be genetically altered cells that have been altered
to overexpress the ATP protein.
[0148] The cells can be cultured under standard culture conditions,
such as those used in Scala et al., (1997) Mol. Pharmacol. 51(6)
1024-33), for MDR or those conditions used in Lee et al., J. Cell.
Biochem. 65(4):513-526 (1997), for MCF-7 AdVp3000.
[0149] The cells are then contacted with a toxic chemotherapy drug,
such as mitoxantrone or daunomycin, in an amount that permits cell
survival due to the resistance conferred by the ATP-binding
cassette protein. The amount used is preferably from about 30 .mu.M
to about 3 mM. The cells are exposed to the drug for a time that is
preferably from about 48 hours to about 96 hours. Cell growth is
measured for these cells based on sulforhodamine staining
measurement. Alternatively, cell growth can be monitored by vital
stains, metabolite measurements or counting cell divisions.
[0150] One specific way to measure cell viability is by a
colorimetric assay (Skehan et al., Natl. Cancer Inst. 82: 1117-1121
(1990)). Cells can be seeded in 96-well plates at 1000 cells/well,
grown for 4 days, and fixed in 50% trichloroacetic acetic acid for
example. The cells can then be stained in 0.4% sulphorhodamine B
dissolved in 1% acetic acid. After washing, the bound dye can be
solubilized with 10 nM unbuffered Tris base, preferably at pH 10.5.
The number of viable cells can then be determined by measuring the
OD at 570 nm. Alternatively, viability of cells can be measured by
counting cells with a cell counter or by incorporation of tritiated
thymidine.
[0151] The cells are then contacted with a compound that inhibits
the biological activity of the ATP-binding cassette protein.
Examples of such an inhibitor include, but are not limited to,
drugs identified as chemosensitizers that are able to restore
sensitivity to cytotoxic agents by inhibiting the transport of PgP
substrates. These include, but are not limited to, calcium channel
antagonists, antiarrhythmics, antihypertensives, diterpenes,
cyclosporines, and many others. The potential inhibitor would be
applied to the cell in an amount from about 1 .mu.M to about 1 mM,
for a time between about 48 and about 72 hours.
[0152] The inhibition of drug resistance can then be detected by
measuring the growth inhibition of cells, using a variety of means,
such as IC50 measurements, vital staining, metabolite measurements,
or confocal microscopy. Confocal microscopy can be used to
determine whether a particular drug has been retained or
accumulated in the cell.
[0153] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0154] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
EXAMPLES
[0155] The following examples are provided by way of illustration
only and not by way of limitation. Those of skill will readily
recognize a variety of noncritical parameters which could be
changed or modified to yield essentially similar results.
Example 1
Cloning of ATP-Binding Cassette Protein MXR1
[0156] cDNA libraries constructed with the SuperScript Plasmid
System for cDNA Synthesis and Plasmid Cloning (GIBCO-BRL, Rockville
Md.) using mRNA from mitoxantrone resistant S1-M1-80 human colon
carcinoma cells can be used to isolate the MXR1 and MXR2 nucleic
acids of the invention.
[0157] The ABC protein (ABCP) gene can be isolated using primers
that flank the coding region of the gene. These primers can be used
to amplify cDNA reverse transcribed from placenta RNA. Primers
corresponding to SEQ ID NO: 5 or 6 can be used to amplify by PCR a
2284 bp product from placenta cDNA. This product can be cloned into
the pGEM-T (Promega Madison, Wis.) vector. The sequence of the
clone can be confirmed by DNA sequencing of the clone using the
original primers as well as 20 nucleotide primers, 300 bp apart,
along the coding region of the ABCP gene.
Example 2
Expression of ATP-Binding Cassette Protein MXR1
[0158] The coding region of the MXR1 gene can be cloned into
appropriate expression vectors to express the gene in cells. This
could be for the purpose of purifying the protein to study its
properties or raise antibodies, or to study the properties of the
protein overexpressed in a mammalian cell line. The full length
coding region of MXR1 can be cloned in the N-terminal to C-terminal
orientation into the pBacPAK8 transfer vector (Clontech, Palo Alto,
Calif.) vector to express the protein in insect cell cultures.
Alternatively the MXR1 gene can be cloned into the pNeoEGFP vector
(Clontech, Palo Alto, Calif.) to express the protein in mammalian
cells. The expression of the protein can be monitored by tagging
the amino or carboxy terminus with an appropriate tag (GFP, his) or
the untagged protein can be monitored using polyclonal or
monoclonal antiserum specific for the MXR1 protein. The nucleotide
and amino acid sequences of MXR1 are provided, respectively, in SEQ
ID NO:1 and 3 and SEQ ID NO:2 and 4.
Example 3
Assay for Identification of Inhibitors of Mitoxantrone
Resistance
[0159] The cells that overexpress MXR1 have been shown to be
resistant to several chemotherapy drugs. These include
mitoxantrone, several anthracyclines, rhodamine, daunomycin, SN-38
(the active metabolite of CPT-11), topotecan, and bisantrene. To
identify compounds that can reverse the effect of MXR1
overexpression, the concentration of drug that inhibits the
proliferation of resistant cells by 50% (IC50) will be measured in
the presence and absence of putative inhibitors. Compounds that
cause a significant decrease in the IC50 will represent inhibitors
and be characterized further. For example, S1-M1-80 cells can be
incubated in media containing 30 micromolar (IC10) of mitoxantrone
and the proliferation of the cells measured by sulforhodamine
staining after 96 hours with and without the addition of various
potential inhibitors, as a screening assay. Next, for compounds
that have potential activity, a formal calculation of the IC50 can
be made by incubating the cells in a range of concentrations,
diluting 3-fold from 3 mM, again, with and without the inhibitor in
question. From this data the IC50 can be calculated. In the absence
of a reversal agent, the IC50 is 100 micromolar for the S1M1-80
cells. Compounds that lower the IC50 for mitoxantrone can be tested
for their ability to also lower the IC50 for other
MXR1-transporting drugs such as adriamycin, topotecan, or
bisantrene.
[0160] As a preliminary screening assay, we can also evaluate the
alteration in accumulation of mitoxantrone by confocal microscopy,
or of rhodamine by FACS analysis. The latter assay, popularized by
investigators working with Pgp antagonists has been used as a
screening tool (see, e.g., Scala, et al., Mol. Pharmacol (1997)
51(6):1024-33). Drug resistant cells are obtained from tissue
culture dishes, plated into each well of a 96 well plate and then
incubated in 1 mM rhodamine 123. Candidate inhibitors are added 15
minutes before addition of the rhodamine. Cells are then incubated
for 1 hour, washed and then resuspended for an efflux period in
medium alone, or medium containing the candidate inhibitor. After
30 minutes, the level of rhodamine remaining in the cells is
tightly correlated with the inhibition of the transporter. A
positive control using energy depletion can be incorporated into
this study.
[0161] Analysis of mitoxantrone accumulation by confocal microscopy
is very straightforward and simple assay, which requires no
preincubation. The experiment can be performed literally under the
microscope, and a 15 minute accumulation of mitoxantrone obtained.
The confocal microscope can be set at a specific sensitivity, and
thus quantitative information gathered when the accumulation is
performed in the presence and absence of the inhibitor.
Sequence CWU 1
1
6 1 2719 DNA Homo sapiens CDS (205)..(2172) MXR1 1 tttaggaacg
caccgtgcac atgcttggtg gtcttgttaa gtggaaactg ctgctttaga 60
gtttgtttgg aaggtccggg tgactcatcc caacatttac atccttaatt gttaaagcgc
120 tgcctccgag cgcacgcatc ctgagatcct gagcctttgg ttaagaccga
gctctattaa 180 gctgaaaaga taaaaactct ccag atg tct tcc agt aat gtc
gaa gtt ttt 231 Met Ser Ser Ser Asn Val Glu Val Phe 1 5 atc cca gtg
tca caa gga aac acc aat ggc ttc ccc gcg aca gtt tcc 279 Ile Pro Val
Ser Gln Gly Asn Thr Asn Gly Phe Pro Ala Thr Val Ser 10 15 20 25 aat
gac ctg aag gca ttt act gaa gga gct gtg tta agt ttt cat aac 327 Asn
Asp Leu Lys Ala Phe Thr Glu Gly Ala Val Leu Ser Phe His Asn 30 35
40 atc tgc tat cga gta aaa ctg aag agt ggc ttt cta cct tgt cga aaa
375 Ile Cys Tyr Arg Val Lys Leu Lys Ser Gly Phe Leu Pro Cys Arg Lys
45 50 55 cca gtt gag aaa gaa ata tta tcg aat atc aat ggg atc atg
aaa cct 423 Pro Val Glu Lys Glu Ile Leu Ser Asn Ile Asn Gly Ile Met
Lys Pro 60 65 70 ggt ctc aac gcc atc ctg gga ccc aca ggt gga ggc
aaa tct tcg tta 471 Gly Leu Asn Ala Ile Leu Gly Pro Thr Gly Gly Gly
Lys Ser Ser Leu 75 80 85 tta gat gtc tta gct gca agg aaa gat cca
agt gga tta tct gga gat 519 Leu Asp Val Leu Ala Ala Arg Lys Asp Pro
Ser Gly Leu Ser Gly Asp 90 95 100 105 gtt ctg ata aat gga gca ccg
cga cct gcc aat ttc aaa tgt aat tca 567 Val Leu Ile Asn Gly Ala Pro
Arg Pro Ala Asn Phe Lys Cys Asn Ser 110 115 120 ggt tac gtg gta caa
gat gat gtt gtg atg ggc act ctg acg gtg aga 615 Gly Tyr Val Val Gln
Asp Asp Val Val Met Gly Thr Leu Thr Val Arg 125 130 135 gaa aac tta
cag ttc tca gca gct ctt cgg ctt gca aca act atg acg 663 Glu Asn Leu
Gln Phe Ser Ala Ala Leu Arg Leu Ala Thr Thr Met Thr 140 145 150 aat
cat gaa aaa aac gaa cgg att aac agg gtc att gaa gag tta ggt 711 Asn
His Glu Lys Asn Glu Arg Ile Asn Arg Val Ile Glu Glu Leu Gly 155 160
165 ctg gat aaa gtg gca gac tcc aag gtt gga act cag ttt atc cgt ggt
759 Leu Asp Lys Val Ala Asp Ser Lys Val Gly Thr Gln Phe Ile Arg Gly
170 175 180 185 gtg tct gga gga gaa aga aaa agg act agt ata gga atg
gag ctt atc 807 Val Ser Gly Gly Glu Arg Lys Arg Thr Ser Ile Gly Met
Glu Leu Ile 190 195 200 act gat cct tcc atc ttg tcc ttg gat gag cct
aca act ggc tta gac 855 Thr Asp Pro Ser Ile Leu Ser Leu Asp Glu Pro
Thr Thr Gly Leu Asp 205 210 215 tca agc aca gca aat gct gtc ctt ttg
ctc ctg aaa agg atg tct aag 903 Ser Ser Thr Ala Asn Ala Val Leu Leu
Leu Leu Lys Arg Met Ser Lys 220 225 230 cag gga cga aca atc atc ttc
tcc att cat cag cct cga tat tcc atc 951 Gln Gly Arg Thr Ile Ile Phe
Ser Ile His Gln Pro Arg Tyr Ser Ile 235 240 245 ttc aag ttg ttt gat
agc ctc acc tta ttg gcc tca gga aga ctt atg 999 Phe Lys Leu Phe Asp
Ser Leu Thr Leu Leu Ala Ser Gly Arg Leu Met 250 255 260 265 ttc cac
ggg cct gct cag gag gcc ttg gga tac ttt gaa tca gct ggt 1047 Phe
His Gly Pro Ala Gln Glu Ala Leu Gly Tyr Phe Glu Ser Ala Gly 270 275
280 tat cac tgt gag gcc tat aat aac cct gca gac ttc ttc ttg gac atc
1095 Tyr His Cys Glu Ala Tyr Asn Asn Pro Ala Asp Phe Phe Leu Asp
Ile 285 290 295 att aat gga gat tcc act gct gtg gca tta aac aga gaa
gaa gac ttt 1143 Ile Asn Gly Asp Ser Thr Ala Val Ala Leu Asn Arg
Glu Glu Asp Phe 300 305 310 aaa gcc aca gag atc ata gag cct tcc aag
cag gat aag cca ctc ata 1191 Lys Ala Thr Glu Ile Ile Glu Pro Ser
Lys Gln Asp Lys Pro Leu Ile 315 320 325 gaa aaa tta gcg gag att tat
gtc aac tcc tcc ttc tac aaa gag aca 1239 Glu Lys Leu Ala Glu Ile
Tyr Val Asn Ser Ser Phe Tyr Lys Glu Thr 330 335 340 345 aaa gct gaa
tta cat caa ctt tcc ggg ggt gag aag aag aag aag atc 1287 Lys Ala
Glu Leu His Gln Leu Ser Gly Gly Glu Lys Lys Lys Lys Ile 350 355 360
aca gtc ttc aag gag atc agc tac acc acc tcc ttc tgt cat caa ctc
1335 Thr Val Phe Lys Glu Ile Ser Tyr Thr Thr Ser Phe Cys His Gln
Leu 365 370 375 aga tgg gtt tcc aag cgt tca ttc aaa aac ttg ctg ggt
aat ccc cag 1383 Arg Trp Val Ser Lys Arg Ser Phe Lys Asn Leu Leu
Gly Asn Pro Gln 380 385 390 gcc tct ata gct cag atc att gtc aca gtc
gta ctg gga ctg gtt ata 1431 Ala Ser Ile Ala Gln Ile Ile Val Thr
Val Val Leu Gly Leu Val Ile 395 400 405 ggt gcc att tac ttt ggg cta
aaa aat gat tct act gga atc cag aac 1479 Gly Ala Ile Tyr Phe Gly
Leu Lys Asn Asp Ser Thr Gly Ile Gln Asn 410 415 420 425 aga gct ggg
gtt ctc ttc ttc ctg acg acc aac cag tgt ttc agc agt 1527 Arg Ala
Gly Val Leu Phe Phe Leu Thr Thr Asn Gln Cys Phe Ser Ser 430 435 440
gtt tca gcc gtg gaa ctc ttt gtg gta gag aag aag ctc ttc ata cat
1575 Val Ser Ala Val Glu Leu Phe Val Val Glu Lys Lys Leu Phe Ile
His 445 450 455 gaa tac atc agc gga tac tac aga gtg tca tct tat ttc
ctt gga aaa 1623 Glu Tyr Ile Ser Gly Tyr Tyr Arg Val Ser Ser Tyr
Phe Leu Gly Lys 460 465 470 ctg tta tct gat tta tta ccc atg agg atg
tta cca agt att ata ttt 1671 Leu Leu Ser Asp Leu Leu Pro Met Arg
Met Leu Pro Ser Ile Ile Phe 475 480 485 acc tgt ata gtg tac ttc atg
tta gga ttg aag cca aag gca gat gcc 1719 Thr Cys Ile Val Tyr Phe
Met Leu Gly Leu Lys Pro Lys Ala Asp Ala 490 495 500 505 ttc ttc gtt
atg atg ttt acc ctt atg atg gtg gct tat tca gcc agt 1767 Phe Phe
Val Met Met Phe Thr Leu Met Met Val Ala Tyr Ser Ala Ser 510 515 520
tcc atg gca ctg gcc ata gca gca ggt cag agt gtg gtt tct gta gca
1815 Ser Met Ala Leu Ala Ile Ala Ala Gly Gln Ser Val Val Ser Val
Ala 525 530 535 aca ctt ctc atg acc atc tgt ttt gtg ttt atg atg att
ttt tca ggt 1863 Thr Leu Leu Met Thr Ile Cys Phe Val Phe Met Met
Ile Phe Ser Gly 540 545 550 ctg ttg gtc aat ctc aca acc att gca tct
tgg ctg tca tgg ctt cag 1911 Leu Leu Val Asn Leu Thr Thr Ile Ala
Ser Trp Leu Ser Trp Leu Gln 555 560 565 tac ttc agc att cca cga tat
gga ttt acg gct ttg cag cat aat gaa 1959 Tyr Phe Ser Ile Pro Arg
Tyr Gly Phe Thr Ala Leu Gln His Asn Glu 570 575 580 585 ttt ttg gga
caa aac ttc tgc cca gga ctc aat gca aca gga aac aat 2007 Phe Leu
Gly Gln Asn Phe Cys Pro Gly Leu Asn Ala Thr Gly Asn Asn 590 595 600
cct tgt aac tat gca aca tgt act ggc gaa gaa tat ttg gta aag cag
2055 Pro Cys Asn Tyr Ala Thr Cys Thr Gly Glu Glu Tyr Leu Val Lys
Gln 605 610 615 ggc atc gat ctc tca ccc tgg ggc ttg tgg aag aat cac
gtg gcc ttg 2103 Gly Ile Asp Leu Ser Pro Trp Gly Leu Trp Lys Asn
His Val Ala Leu 620 625 630 gct tgt atg att gtt att ttc ctc aca att
gcc tac ctg aaa ttg tta 2151 Ala Cys Met Ile Val Ile Phe Leu Thr
Ile Ala Tyr Leu Lys Leu Leu 635 640 645 ttt ctt aaa aaa tat tct
taaatttccc cttaattcag tatgatttat 2199 Phe Leu Lys Lys Tyr Ser 650
655 cctcacataa aaaagaagca ctttgattga agtattcaat caagtttttt
tgttgttttc 2259 tgttcccttg ccatcacact gttgcacagc agcaattgtt
ttaaagagat acatttttag 2319 aaatcacaac aaactgaatt aaacatgaaa
gaacccaaga catcatgtat cgcatattag 2379 ttaatctcct cagacagtaa
ccatggggaa gaaatctggt ctaatttatt aatctaaaaa 2439 aggagaattg
aattctggaa actcctgaca agttattact gtctctggca tttgtttcct 2499
catctttaaa atgaataggt aggttagtag cccttcagtc ttaatacttt atgatgctat
2559 ggtttgccat tatttaatat atgacaaatg tattaatgct atactggaaa
tgtaaaattg 2619 aaaatatgtt ggaaaaaaga ttctgtctta tagggtaaaa
aaagccaccg gtgatagaaa 2679 aaaaatcttt ttgataagca cattaaagtt
aatagaactt 2719 2 655 PRT Homo sapiens 2 Met Ser Ser Ser Asn Val
Glu Val Phe Ile Pro Val Ser Gln Gly Asn 1 5 10 15 Thr Asn Gly Phe
Pro Ala Thr Val Ser Asn Asp Leu Lys Ala Phe Thr 20 25 30 Glu Gly
Ala Val Leu Ser Phe His Asn Ile Cys Tyr Arg Val Lys Leu 35 40 45
Lys Ser Gly Phe Leu Pro Cys Arg Lys Pro Val Glu Lys Glu Ile Leu 50
55 60 Ser Asn Ile Asn Gly Ile Met Lys Pro Gly Leu Asn Ala Ile Leu
Gly 65 70 75 80 Pro Thr Gly Gly Gly Lys Ser Ser Leu Leu Asp Val Leu
Ala Ala Arg 85 90 95 Lys Asp Pro Ser Gly Leu Ser Gly Asp Val Leu
Ile Asn Gly Ala Pro 100 105 110 Arg Pro Ala Asn Phe Lys Cys Asn Ser
Gly Tyr Val Val Gln Asp Asp 115 120 125 Val Val Met Gly Thr Leu Thr
Val Arg Glu Asn Leu Gln Phe Ser Ala 130 135 140 Ala Leu Arg Leu Ala
Thr Thr Met Thr Asn His Glu Lys Asn Glu Arg 145 150 155 160 Ile Asn
Arg Val Ile Glu Glu Leu Gly Leu Asp Lys Val Ala Asp Ser 165 170 175
Lys Val Gly Thr Gln Phe Ile Arg Gly Val Ser Gly Gly Glu Arg Lys 180
185 190 Arg Thr Ser Ile Gly Met Glu Leu Ile Thr Asp Pro Ser Ile Leu
Ser 195 200 205 Leu Asp Glu Pro Thr Thr Gly Leu Asp Ser Ser Thr Ala
Asn Ala Val 210 215 220 Leu Leu Leu Leu Lys Arg Met Ser Lys Gln Gly
Arg Thr Ile Ile Phe 225 230 235 240 Ser Ile His Gln Pro Arg Tyr Ser
Ile Phe Lys Leu Phe Asp Ser Leu 245 250 255 Thr Leu Leu Ala Ser Gly
Arg Leu Met Phe His Gly Pro Ala Gln Glu 260 265 270 Ala Leu Gly Tyr
Phe Glu Ser Ala Gly Tyr His Cys Glu Ala Tyr Asn 275 280 285 Asn Pro
Ala Asp Phe Phe Leu Asp Ile Ile Asn Gly Asp Ser Thr Ala 290 295 300
Val Ala Leu Asn Arg Glu Glu Asp Phe Lys Ala Thr Glu Ile Ile Glu 305
310 315 320 Pro Ser Lys Gln Asp Lys Pro Leu Ile Glu Lys Leu Ala Glu
Ile Tyr 325 330 335 Val Asn Ser Ser Phe Tyr Lys Glu Thr Lys Ala Glu
Leu His Gln Leu 340 345 350 Ser Gly Gly Glu Lys Lys Lys Lys Ile Thr
Val Phe Lys Glu Ile Ser 355 360 365 Tyr Thr Thr Ser Phe Cys His Gln
Leu Arg Trp Val Ser Lys Arg Ser 370 375 380 Phe Lys Asn Leu Leu Gly
Asn Pro Gln Ala Ser Ile Ala Gln Ile Ile 385 390 395 400 Val Thr Val
Val Leu Gly Leu Val Ile Gly Ala Ile Tyr Phe Gly Leu 405 410 415 Lys
Asn Asp Ser Thr Gly Ile Gln Asn Arg Ala Gly Val Leu Phe Phe 420 425
430 Leu Thr Thr Asn Gln Cys Phe Ser Ser Val Ser Ala Val Glu Leu Phe
435 440 445 Val Val Glu Lys Lys Leu Phe Ile His Glu Tyr Ile Ser Gly
Tyr Tyr 450 455 460 Arg Val Ser Ser Tyr Phe Leu Gly Lys Leu Leu Ser
Asp Leu Leu Pro 465 470 475 480 Met Arg Met Leu Pro Ser Ile Ile Phe
Thr Cys Ile Val Tyr Phe Met 485 490 495 Leu Gly Leu Lys Pro Lys Ala
Asp Ala Phe Phe Val Met Met Phe Thr 500 505 510 Leu Met Met Val Ala
Tyr Ser Ala Ser Ser Met Ala Leu Ala Ile Ala 515 520 525 Ala Gly Gln
Ser Val Val Ser Val Ala Thr Leu Leu Met Thr Ile Cys 530 535 540 Phe
Val Phe Met Met Ile Phe Ser Gly Leu Leu Val Asn Leu Thr Thr 545 550
555 560 Ile Ala Ser Trp Leu Ser Trp Leu Gln Tyr Phe Ser Ile Pro Arg
Tyr 565 570 575 Gly Phe Thr Ala Leu Gln His Asn Glu Phe Leu Gly Gln
Asn Phe Cys 580 585 590 Pro Gly Leu Asn Ala Thr Gly Asn Asn Pro Cys
Asn Tyr Ala Thr Cys 595 600 605 Thr Gly Glu Glu Tyr Leu Val Lys Gln
Gly Ile Asp Leu Ser Pro Trp 610 615 620 Gly Leu Trp Lys Asn His Val
Ala Leu Ala Cys Met Ile Val Ile Phe 625 630 635 640 Leu Thr Ile Ala
Tyr Leu Lys Leu Leu Phe Leu Lys Lys Tyr Ser 645 650 655 3 502 DNA
Mus sp. CDS (1)..(444) MXR1 3 ttc ggc cta ggg gcc gag gct tat acg
gcc agt tcc atg gca ctg gcc 48 Phe Gly Leu Gly Ala Glu Ala Tyr Thr
Ala Ser Ser Met Ala Leu Ala 1 5 10 15 ata gcc aca ggc caa agt gtg
gtg tct gta gca aca cta ctc atg aca 96 Ile Ala Thr Gly Gln Ser Val
Val Ser Val Ala Thr Leu Leu Met Thr 20 25 30 atc gct ttt gta ttt
atg atg ctc ttt tct ggc ctc ttg gtg aat ctc 144 Ile Ala Phe Val Phe
Met Met Leu Phe Ser Gly Leu Leu Val Asn Leu 35 40 45 aga acc att
ggg cct tgg ctg tcc tgg ctt cag tac ttt agc att cct 192 Arg Thr Ile
Gly Pro Trp Leu Ser Trp Leu Gln Tyr Phe Ser Ile Pro 50 55 60 cga
tat ggc ttc aca gct ttg cag tat aat gaa ttc ttg gga caa gag 240 Arg
Tyr Gly Phe Thr Ala Leu Gln Tyr Asn Glu Phe Leu Gly Gln Glu 65 70
75 80 ttt tgt cca gga ttc aat gta acg gac aac agc act tgt gtt aac
agc 288 Phe Cys Pro Gly Phe Asn Val Thr Asp Asn Ser Thr Cys Val Asn
Ser 85 90 95 tat gca ata tgt act ggt aac gag tac ttg ata aat cag
ggc atc gaa 336 Tyr Ala Ile Cys Thr Gly Asn Glu Tyr Leu Ile Asn Gln
Gly Ile Glu 100 105 110 ctg tca cct tgg gga ctg tgg aag aat cat gtg
gcc ctg gct tgt atg 384 Leu Ser Pro Trp Gly Leu Trp Lys Asn His Val
Ala Leu Ala Cys Met 115 120 125 att att atc ttc ctc aca att gcc tac
ctg aaa ttg ttg ttt ctt aaa 432 Ile Ile Ile Phe Leu Thr Ile Ala Tyr
Leu Lys Leu Leu Phe Leu Lys 130 135 140 aag tat tct taatttcccc
tttaacggac tattaattgt actccaatta 481 Lys Tyr Ser 145 aatatgggca
ctttgattac c 502 4 147 PRT Mus sp. 4 Phe Gly Leu Gly Ala Glu Ala
Tyr Thr Ala Ser Ser Met Ala Leu Ala 1 5 10 15 Ile Ala Thr Gly Gln
Ser Val Val Ser Val Ala Thr Leu Leu Met Thr 20 25 30 Ile Ala Phe
Val Phe Met Met Leu Phe Ser Gly Leu Leu Val Asn Leu 35 40 45 Arg
Thr Ile Gly Pro Trp Leu Ser Trp Leu Gln Tyr Phe Ser Ile Pro 50 55
60 Arg Tyr Gly Phe Thr Ala Leu Gln Tyr Asn Glu Phe Leu Gly Gln Glu
65 70 75 80 Phe Cys Pro Gly Phe Asn Val Thr Asp Asn Ser Thr Cys Val
Asn Ser 85 90 95 Tyr Ala Ile Cys Thr Gly Asn Glu Tyr Leu Ile Asn
Gln Gly Ile Glu 100 105 110 Leu Ser Pro Trp Gly Leu Trp Lys Asn His
Val Ala Leu Ala Cys Met 115 120 125 Ile Ile Ile Phe Leu Thr Ile Ala
Tyr Leu Lys Leu Leu Phe Leu Lys 130 135 140 Lys Tyr Ser 145 5 20
DNA Artificial Sequence Description of Artificial SequencePCR
primer ABCPF1 5 acgcaccgtg cacatgcttg 20 6 23 DNA Artificial
Sequence Description of Artificial SequencePCR primer ABCPR1 6
acagtgtgat ggcaagggaa cag 23
* * * * *
References