U.S. patent application number 10/435324 was filed with the patent office on 2003-09-25 for insulin receptor tyrosine kinase substrate.
This patent application is currently assigned to Incyte Corporation. Invention is credited to Baughn, Mariah R., Corley, Neil C., Guegler, Karl J., Hillman, Jennifer L..
Application Number | 20030180810 10/435324 |
Document ID | / |
Family ID | 25372286 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030180810 |
Kind Code |
A1 |
Hillman, Jennifer L. ; et
al. |
September 25, 2003 |
Insulin receptor tyrosine kinase substrate
Abstract
The invention provides a human insulin receptor tyrosine kinase
substrate (IRS-p53h) and polynucleotides which identify and encode
IRS-p53h. The invention also provides expression vectors, host
cells, antibodies, agonists, and antagonists. The invention also
provides methods for treating or preventing disorders associated
with expression of IRS-p53h.
Inventors: |
Hillman, Jennifer L.; (Santa
Cruz, CA) ; Corley, Neil C.; (Castro Valley, CA)
; Guegler, Karl J.; (Menlo Park, CA) ; Baughn,
Mariah R.; (San Leandro, CA) |
Correspondence
Address: |
INCYTE CORPORATION (formerly known as Incyte
Genomics, Inc.)
3160 PORTER DRIVE
PALO ALTO
CA
94304
US
|
Assignee: |
Incyte Corporation
3160 Porter Drive
Palo Alto
CA
94304
|
Family ID: |
25372286 |
Appl. No.: |
10/435324 |
Filed: |
May 9, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10435324 |
May 9, 2003 |
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09046572 |
Mar 23, 1998 |
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6589935 |
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09046572 |
Mar 23, 1998 |
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08878563 |
Jun 19, 1997 |
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5891674 |
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Current U.S.
Class: |
435/7.1 ;
435/320.1; 435/328; 435/70.21; 530/387.3; 536/23.53 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 35/00 20180101; A61K 38/00 20130101; A61P 3/10 20180101; C07K
14/47 20130101 |
Class at
Publication: |
435/7.1 ;
435/70.21; 435/328; 435/320.1; 530/387.3; 536/23.53 |
International
Class: |
G01N 033/53; C07H
021/04; C12P 021/04; C12N 005/06; C07K 016/44 |
Claims
What is claimed is:
1. An isolated antibody which specifically binds to a polypeptide
comprising an amino acid sequence of SEQ ID NO:3.
2. The antibody of claim 1, wherein the antibody is: a) a chimeric
antibody, b) a single chain antibody, c) a Fab fragment, d) a
F(ab').sub.2 fragment, or e) a humanized antibody.
3. A composition comprising an antibody of claim 1 and an
acceptable excipient.
4. A method of diagnosing a condition or disease associated with
the expression of IRS-p53h-2 in a subject, comprising administering
to said subject an effective amount of the composition of claim
3.
5. A composition of claim 3, wherein the antibody is labeled.
6. A method of diagnosing a condition or disease associated with
the expression of IRS-p53h-2 in a subject, comprising administering
to said subject an effective amount of the composition of claim
5.
7. A method of preparing a polyclonal antibody with the specificity
of the antibody of claim 1, the method comprising: a) immunizing an
animal with a polypeptide consisting of an amino acid sequence of
SEQ ID NO:3, or an immunogenic fragment thereof, under conditions
to elicit an antibody response, b) isolating antibodies from said
animal, and c) screening the isolated antibodies with the
polypeptide, thereby identifying a polyclonal antibody which binds
specifically to a polypeptide comprising an amino acid sequence of
SEQ ID NO:3.
8. A polyclonal antibody produced by a method of claim 7.
9. A composition comprising the polyclonal antibody of claim 8 and
a suitable carrier.
10. A method of making a monoclonal antibody with the specificity
of the antibody of claim 1, the method comprising: a) immunizing an
animal with a polypeptide consisting of an amino acid sequence of
SEQ ID NO:3, or an immunogenic fragment thereof, under conditions
to elicit an antibody response, b) isolating antibody producing
cells from the animal, c) fusing the antibody producing cells with
immortalized cells to form monoclonal antibody-producing hybridoma
cells, d) culturing the hybridoma cells, and e) isolating from the
culture monoclonal antibody which binds specifically to a
polypeptide comprising an amino acid sequence of SEQ ID NO:3.
11. A monoclonal antibody produced by a method of claim 10.
12. A composition comprising the monoclonal antibody of claim 11
and a suitable carrier.
13. The antibody of claim 1, wherein the antibody is produced by
screening a Fab expression library.
14. The antibody of claim 1, wherein the antibody is produced by
screening a recombinant immunoglobulin library.
15. A method of detecting a polypeptide comprising an amino acid
sequence of SEQ ID NO:3 in a sample, the method comprising: a)
incubating the antibody of claim 11 with a sample under conditions
to allow specific binding of the antibody and the polypeptide, and
b) detecting specific binding, wherein specific binding indicates
the presence of a polypeptide comprising an amino acid sequence of
SEQ ID NO:3 in the sample.
16. A method of purifying a polypeptide comprising an amino acid
sequence of SEQ ID NO:3 from a sample, the method comprising: a)
incubating the antibody of claim 1 with a sample under conditions
to allow specific binding of the antibody and the polypeptide, and
b) separating the antibody from the sample and obtaining the
purified polypeptide comprising an amino acid sequence of SEQ ID
NO:3.
17. The method of claim 5, wherein the disease or condition is a
disorder of the insulin response and is selected from the group
consisting of type 2 (non-insulin-dependent) diabetes mellitus,
hyperglycemia, myotonic muscular dystrophy, acanthosis nigricans,
retinopathy, nephropathy, atherosclerotic coronary and peripheral
arterial disease, and peripheral and autonomic neuropathies.
18. The method of claim 6 wherein the disease or condition is
Alzheimer's disease.
19. A method of treating or preventing a disease or condition
associated with increased expression of IRS-p53h-2, the method
comprising administering to a subject in need of such treatment the
composition of claim 9.
20. A method of treating or preventing a disease or condition
associated with increasedd expression of IRS-p53h-2, the method
comprising administering to a subject in need of such treatment the
composition of claim 12.
Description
[0001] This application is a divisional application of copending
U.S. application Ser. No. 09/046,572, filed Mar. 23, 1998, which is
a continuation-in-part of U.S. application Ser. No. 08/878,563,
filed Jun. 19, 1997, now U.S. Pat. No. 5,891,674.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of two human insulin receptor tyrosine kinase substrates
and to the use of these sequences in the diagnosis, prevention, and
treatment of reproductive disorders, Alzheimer's disease, cancer,
immunological disorders, and disorders associated with insulin
response.
BACKGROUND OF THE INVENTION
[0003] Insulin controls blood glucose levels by stimulating glucose
influx and metabolism in muscle and adipocytes and by inhibiting
gluconeogenesis in the liver. Insulin also modifies the expression
or the activity of a variety of enzymes and transport systems in
nearly all cells.
[0004] Insulin action is mediated through the insulin receptor
(IR), a transmembrane glycoprotein with protein tyrosine kinase
(PTK) activity. Insulin binding triggers receptor
autophosphorylation which activates PTK activity. The cellular
response to insulin is mediated through tyrosine phosphorylation of
cytosolic polypeptide substrates which act as second messengers in
IR signal transduction. Once phosphorylated, the substrates bind to
and activate various signal transduction proteins. The signal
transduction proteins contain Src-homology-2 (SH2)-domains which
bind phosphotyrosine-containing peptide motifs.
[0005] Several IR-PTK substrates have been described. The most
extensively characterized substrate is the 185-kDa insulin receptor
substrate-1 (IRS-1). IRS-1 is found in a variety of insulin
responsive cells and tissues. It exhibits no intrinsic enzyme
activity but, once phosphorylated, binds to and activates
SH2-containing signal transduction proteins including
phosphatidylinositol (PI) 3'-kinase and GRB-2, a regulator of the
Ras pathway. (White, M. F. et al. (1994) J. Biol. Chem. 269:1-4.)
Mutations in the IRS-1 gene impairs insulin-stimulated signaling
and may contribute to insulin resistance in normal and diabetic
populations. (Almind, K. et al. (1996) J. Clin. Invest.
97:2569-2575.)
[0006] Two 60-kDa protein substrates of the IR-PTK have been
identified. One associates with the GTPase activator of Ras (termed
GAP) and the other associates with PI 3'-kinase. (Yeh, T. et al.
(1996) J. Biol. Chem. 271:2921-2928.) Two additional substrates for
IR-PTK with molecular masses of 53 and 58 kDa were recently
identified in rodents. These proteins, p53 and p58, are closely
related and may arise from alternative splicing of mRNA or
differential post-translational modifications. P53 and p58 do not
associate with GAP or PI 3'-kinase and are immunologically distinct
from the 60-kDa GAP-associated protein and the 60-kDa PI
3'-kinase-associated protein. P53 contains a microbodies C-terminal
targeting signal which enables import of the protein into
peroxisomes, glyoxysomes, and glycosomes. (Yeh, et al., supra.)
[0007] Post-receptor defects in the insulin signaling pathway are a
common feature of type 2 (non-insulin-dependent) diabetes mellitus.
(Stoffel M. et al. (1993) Diabetologia 36: 335-337.) Other
disorders or conditions associated with disturbances in insulin
response include hyperglycemia, myotonic muscular dystrophy,
acanthosis nigricans, retinopathy, nephropathy, atherosclerotic
coronary and peripheral arterial disease, and peripheral and
autonomic neuropathies.
[0008] The discovery of two new human insulin receptor tyrosine
kinase substrates and the polynucleotides encoding them satisfies a
need in the art by providing new compositions which are useful in
the diagnosis, prevention and treatment of reproductive disorders,
Alzheimer's disease, cancer, immunological disorders, and disorders
associated with insulin response.
SUMMARY OF THE INVENTION
[0009] The invention features substantially purified polypeptides
comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 or
a fragment of SEQ ID NO:1 or SEQ ID NO:3.
[0010] The invention further provides a substantially purified
variant having at least 90% amino acid sequence identity to the
amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:3 or a fragment of
SEQ ID NO:1 or SEQ ID NO:3. The invention also provides an isolated
and purified polynucleotide encoding the polypeptide comprising the
sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1
or SEQ ID NO:3. The invention also includes an isolated and
purified polynucleotide variant having at least 90% polynucleotide
sequence identity to the polynucleotide encoding the polypeptide
comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 or
a fragment of SEQ ID NO:1 or SEQ ID NO:3.
[0011] The invention further provides an isolated and purified
polynucleotide which hybridizes under stringent conditions to the
polynucleotide encoding the polypeptide comprising the amino acid
sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1
or SEQ ID NO:3, as well as an isolated and purified polynucleotide
which is complementary to the polynucleotide encoding the
polypeptide comprising the amino acid sequence of SEQ ID NO:1 or
SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3.
[0012] The invention also provides an isolated and purified
polynucleotide comprising the polynucleotide sequence of SEQ ID
NO:2 and SEQ ID NO:4 or a fragment of SEQ ID NO:2 and SEQ ID NO:4,
and an isolated and purified polynucleotide variant having at least
90% polynucleotide sequence identity to the polynucleotide
comprising the polynucleotide sequence of SEQ ID NO:2 and SEQ ID
NO:4 or a fragment of SEQ ID NO:2 and SEQ ID NO:4. The invention
also provides an isolated and purified polynucleotide having a
sequence complementary to the polynucleotide comprising the
polynucleotide sequence of SEQ ID NO:2 and SEQ ID NO:4 or a
fragment of SEQ ID NO:2 and SEQ ID NO:4.
[0013] The invention further provides an expression vector
containing at least a fragment of the polynucleotide encoding the
polypeptide comprising the sequence of SEQ ID NO:1 or SEQ ID NO:3
or a fragment of SEQ ID NO:1 or SEQ ID NO:3. In another aspect, the
expression vector is contained within a host cell.
[0014] The invention also provides a method for producing a
polypeptide comprising the amino acid sequence of SEQ ID NO:1 or
SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3, the method
comprising the steps of: (a) culturing the host cell containing an
expression vector containing at least a fragment of a
polynucleotide encoding the polypeptide comprising the amino acid
sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1
or SEQ ID NO:3 under conditions suitable for the expression of the
polypeptide; and (b) recovering the polypeptide from the host cell
culture.
[0015] The invention also provides a pharmaceutical composition
comprising a substantially purified polypeptide having the sequence
of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ
ID NO:3 in conjunction with a suitable pharmaceutical carrier.
[0016] The invention further includes a purified antibody which
binds to a polypeptide comprising the sequence of SEQ ID NO:1 or
SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID NO:3, as well as
a purified agonist and a purified antagonist of the
polypeptide.
[0017] The invention also provides a method for treating or
preventing a disorder associated with insulin response, the method
comprising administering to a subject in need of such treatment an
effective amount of a pharmaceutical composition comprising
substantially purified polypeptide having the amino acid sequence
of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ
ID NO:3.
[0018] The invention also provides a method for treating or
preventing a reproductive disorder, the method comprising
administering to a subject in need of such treatment an effective
amount of an antagonist of the polypeptide having the amino acid
sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1
or SEQ ID NO:3.
[0019] The invention also provides a method for treating or
preventing Alzheimer's disease, the method comprising administering
to a subject in need of such treatment an effective amount of an
antagonist of the polypeptide having the amino acid sequence of SEQ
ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID
NO:3.
[0020] The invention also provides a method for treating or
preventing a cancer, the method comprising administering to a
subject in need of such treatment an effective amount of an
antagonist of the polypeptide having the amino acid sequence of SEQ
ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1 or SEQ ID
NO:3.
[0021] The invention also provides a method for treating or
preventing an immunological disorder, the method comprising
administering to a subject in need of such treatment an effective
amount of an antagonist of the polypeptide having the amino acid
sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1
or SEQ ID NO:3.
[0022] The invention also provides a method for detecting a
polynucleotide encoding a polypeptide comprising the amino acid
sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1
or SEQ ID NO:3 in a biological sample containing nucleic acids, the
method comprising the steps of: (a) hybridizing the complement of
the polynucleotide encoding the polypeptide comprising the amino
acid sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID
NO:1 or SEQ ID NO:3 to at least one of the nucleic acids of the
biological sample, thereby forming a hybridization complex; and (b)
detecting the hybridization complex, wherein the presence of the
hybridization complex correlates with the presence of a
polynucleotide encoding the polypeptide comprising the amino acid
sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment of SEQ ID NO:1
or SEQ ID NO:3 in the biological sample. In one aspect, the nucleic
acids of the biological sample are amplified by the polymerase
chain reaction prior to the hybridizing step.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIGS. 1A, 1B, 1C, 1D, 1E, and 1F show the amino acid
sequence (SEQ ID NO:1) and nucleic acid sequence (SEQ ID NO:2) of
IRS-p53h-1. The alignment was produced using MacDNASIS PRO.TM.
software (Hitachi Software Engineering Co. Ltd. San Bruno,
Calif.).
[0024] FIGS. 2A, 2B, 2C, 2D, 2E, and 2F show the amino acid
sequence (SEQ ID NO:3) and nucleic acid sequence (SEQ ID NO:4) of
IRS-p53h-2. The alignment was produced using MacDNASIS PRO.TM.
software (Hitachi Software Engineering Co. Ltd., San Bruno,
Calif.).
[0025] FIGS. 3A, 3B, 3C, and 3D show the amino acid sequence
alignments among IRS-p53h-1 (918158; SEQ ID NO:1), IRS-p53h-2
(2493150; SEQ ID NO:3), and IRS p53 from hamster (GI 1203820; SEQ
ID NO:5), produced using the multisequence alignment program of
LASERGENE.TM. software (DNASTAR Inc, Madison Wis.).
DESCRIPTION OF THE INVENTION
[0026] Before the present proteins, nucleotide sequences, and
methods are described, it is understood that this invention is not
limited to the particular methodology, protocols, cell lines,
vectors, and reagents described, as these may vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
limit the scope of the present invention which will be limited only
by the appended claims.
[0027] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, a reference to "a host cell" includes a plurality of such
host cells, and a reference to "an antibody" is a reference to one
or more antibodies and equivalents thereof known to those skilled
in the art, and so forth.
[0028] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices, and materials are now
described. All publications mentioned herein are cited for the
purpose of describing and disclosing the cell lines, vectors, and
methodologies which are reported in the publications and which
might be used in connection with the invention. Nothing herein is
to be construed as an admission that the invention is not entitled
to antedate such disclosure by virtue of prior invention.
DEFINITIONS
[0029] "IRS-p53h," as used herein, refers to the amino acid
sequences of substantially purified IRS-p53h obtained from any
species, particularly a mammalian species, including bovine, ovine,
porcine, murine, equine, and preferably the human species, from any
source, whether natural, synthetic, semi-synthetic, or
recombinant.
[0030] The term "agonist," as used herein, refers to a molecule
which, when bound to IRS-p53h, increases or prolongs the duration
of the effect of IRS-p53h. Agonists may include proteins, nucleic
acids, carbohydrates, or any other molecules which bind to and
modulate the effect of IRS-p53h.
[0031] An "allele" or an "allelic sequence," as these terms are
used herein, is an alternative form of the gene encoding IRS-p53h.
Alleles may result from at least one mutation in the nucleic acid
sequence and may result in altered mRNAs or in polypeptides whose
structure or function may or may not be altered. Any given natural
or recombinant gene may have none, one, or many allelic forms.
Common mutational changes which give rise to alleles are generally
ascribed to natural deletions, additions, or substitutions of
nucleotides. Each of these types of changes may occur alone, or in
combination with the others, one or more times in a given
sequence.
[0032] "Altered" nucleic acid sequences encoding IRS-p53h, as
described herein, include those sequences with deletions,
insertions, or substitutions of different nucleotides, resulting in
a polynucleotide the same IRS-p53h or a polypeptide with at least
one functional characteristic of IRS-p53h. Included within this
definition are polymorphisms which may or may not be readily
detectable using a particular oligonucleotide probe of the
polynucleotide encoding IRS-p53h, and improper or unexpected
hybridization to alleles, with a locus other than the normal
chromosomal locus for the polynucleotide sequence encoding
IRS-p53h. The encoded protein may also be "altered," and may
contain deletions, insertions, or substitutions of amino acid
residues which produce a silent change and result in a functionally
equivalent IRS-p53h. Deliberate amino acid substitutions may be
made on the basis of similarity in polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues, as long as the biological or immunological activity
of IRS-p53h is retained. For example, negatively charged amino
acids may include aspartic acid and glutamic acid, positively
charged amino acids may include lysine and arginine, and amino
acids with uncharged polar head groups having similar
hydrophilicity values may include leucine, isoleucine, and valine;
glycine and alanine; asparagine and glutamine; serine and
threonine; and phenylalanine and tyrosine.
[0033] The terms "amino acid" or "amino acid sequence," as used
herein, refer to an oligopeptide, peptide, polypeptide, or protein
sequence, or a fragment of any of these, and to naturally occurring
or synthetic molecules. In this context, "fragments", "immunogenic
fragments", or "antigenic fragments" refer to fragments of IRS-p53h
which are preferably about 5 to about 15 amino acids in length and
which retain some biological activity or immunological activity of
IRS-p53h. Where "amino acid sequence" is recited herein to refer to
an amino acid sequence of a naturally occurring protein molecule,
"amino acid sequence" and like terms are not meant to limit the
amino acid sequence to the complete native amino acid sequence
associated with the recited protein molecule.
[0034] "Amplification," as used herein, relates to the production
of additional copies of a nucleic acid sequence. Amplification is
generally carried out using polymerase chain reaction (PCR)
technologies well known in the art. (See, e.g., Dieffenbach, C. W.
and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold
Spring Harbor Press, Plainview, N.Y., pp.1-5.)
[0035] The term "antagonist," as it is used herein, refers to a
molecule which, when bound to IRS-p53h, decreases the amount or the
duration of the effect of the biological or immunological activity
of IRS-p53h. Antagonists may include proteins, nucleic acids,
carbohydrates, antibodies, or any other molecules which decrease
the effect of IRS-p53h.
[0036] As used herein, the term "antibody" refers to intact
molecules as well as to fragments thereof, such as Fa,
F(ab').sub.2, and Fv fragments, which are capable of binding the
epitopic determinant. Antibodies that bind IRS-p53h polypeptides
can be prepared using intact polypeptides or using fragments
containing small peptides of interest as the immunizing antigen.
The polypeptide or oligopeptide used to immunize an animal (e.g., a
mouse, a rat, or a rabbit) can be derived from the translation of
RNA, or synthesized chemically, and can be conjugated to a carrier
protein if desired. Commonly used carriers that are chemically
coupled to peptides include bovine serum albumin, thyroglobulin,
and keyhole limpet hemocyanin (KLH). The coupled peptide is then
used to immunize the animal.
[0037] The term "antigenic determinant," as used herein, refers to
that fragment of a molecule (i.e., an epitope) that makes contact
with a particular antibody. When a protein or a fragment of a
protein is used to immunize a host animal, numerous regions of the
protein may induce the production of antibodies which bind
specifically to antigenic determinants (given regions or
three-dimensional structures on the protein). An antigenic
determinant may compete with the intact antigen (i.e., the
immunogen used to elicit the immune response) for binding to an
antibody.
[0038] The term "antisense," as used herein, refers to any
composition containing a nucleic acid sequence which is
complementary to a specific nucleic acid sequence. The term
"antisense strand" is used in reference to a nucleic acid strand
that is complementary to the "sense" strand. Antisense molecules
may be produced by any method including synthesis or transcription.
Once introduced into a cell, the complementary nucleotides combine
with natural sequences produced by the cell to form duplexes and to
block either transcription or translation. The designation
"negative" can refer to the antisense strand, and the designation
"positive" can refer to the sense strand.
[0039] As used herein, the term "biologically active," refers to a
protein having structural, regulatory, or biochemical functions of
a naturally occurring molecule. Likewise, "immunologically active"
refers to the capability of the natural, recombinant, or synthetic
IRS-p53h, or of any oligopeptide thereof, to induce a specific
immune response in appropriate animals or cells and to bind with
specific antibodies.
[0040] The terms "complementary" or "complementarity," as used
herein, refer to the natural binding of polynucleotides under
permissive salt and temperature conditions by base pairing. For
example, the sequence "A-G-T" binds to the complementary sequence
"T-C-A." Complementarity between two single-stranded molecules may
be "partial," such that only some of the nucleic acids bind, or it
may be "complete," such that total complementarity exists between
the single stranded molecules. The degree of complementarity
between nucleic acid strands has significant effects on the
efficiency and strength of the hybridization between the nucleic
acid strands. This is of particular importance in amplification
reactions, which depend upon binding between nucleic acids strands,
and in the design and use of peptide nucleic acid (PNA)
molecules.
[0041] A "composition comprising a given polynucleotide sequence"
or a "composition comprising a given amino acid sequence," as these
terms are used herein, refer broadly to any composition containing
the given polynucleotide or amino acid sequence. The composition
may comprise a dry formulation, an aqueous solution, or a sterile
composition. Compositions comprising polynucleotide sequences
encoding IRS-p53h or fragments of IRS-p53h may be employed as
hybridization probes. The probes may be stored in freeze-dried form
and may be associated with a stabilizing agent such as a
carbohydrate. In hybridizations, the probe may be deployed in an
aqueous solution containing salts (e.g., NaCl), detergents (e.g.,
SDS), and other components (e.g., Denhardt's solution, dry milk,
salmon sperm DNA, etc.).
[0042] "Consensus sequence," as used herein, refers to a nucleic
acid sequence which has been resequenced to resolve uncalled bases,
extended using XL-PCR.TM. (Perkin Elmer, Norwalk, Conn.) in the 5'
and/or the 3' direction, and resequenced, or which has been
assembled from the overlapping sequences of more than one Incyte
Clone using a computer program for fragment assembly, such as the
GELVIEW.TM. Fragment Assembly system (GCG, Madison, Wis.). Some
sequences have been both extended and assembled to produce the
consensus sequence.
[0043] As used herein, the term "correlates with expression of a
polynucleotide" indicates that the detection of the presence of
nucleic acids, the same or related to a nucleic acid sequence
encoding IRS-p53h, by northern analysis is indicative of the
presence of nucleic acids encoding IRS-p53h in a sample, and
thereby correlates with expression of the transcript from the
polynucleotide encoding IRS-p53h.
[0044] A "deletion," as the term is used herein, refers to a change
in the amino acid or nucleotide sequence that results in the
absence of one or more amino acid residues or nucleotides.
[0045] The term "derivative," as used herein, refers to the
chemical modification of IRS-p53h, of a polynucleotide sequence
encoding IRS-p53h, or of a polynucleotide sequence complementary to
a polynucleotide sequence encoding IRS-p53h. Chemical modifications
of a polynucleotide sequence can include, for example, replacement
of hydrogen by an alkyl, acyl, or amino group. A derivative
polynucleotide encodes a polypeptide which retains at least one
biological or immunological function of the natural molecule. A
derivative polypeptide is one modified by glycosylation,
pegylation, or any similar process that retains at least one
biological or immunological function of the polypeptide from which
it was derived.
[0046] The term "homology," as used herein, refers to a degree of
complementarity. There may be partial homology or complete
homology. The word "identity" may substitute for the word
"homology." A partially complementary sequence that at least
partially inhibits an identical sequence from hybridizing to a
target nucleic acid is referred to as "substantially homologous."
The inhibition of hybridization of the completely complementary
sequence to the target sequence may be examined using a
hybridization assay (Southern or northern blot, solution
hybridization, and the like) under conditions of reduced
stringency. A substantially homologous sequence or hybridization
probe will compete for and inhibit the binding of a completely
homologous sequence to the target sequence under conditions of
reduced stringency. This is not to say that conditions of reduced
stringency are such that non-specific binding is permitted, as
reduced stringency conditions require that the binding of two
sequences to one another be a specific (i.e., a selective)
interaction. The absence of non-specific binding may be tested by
the use of a second target sequence which lacks even a partial
degree of complementarity (e.g., less than about 30% homology or
identity). In the absence of non-specific binding, the
substantially homologous sequence or probe will not hybridize to
the second non-complementary target sequence.
[0047] The phrases "percent identity" or "% identity" refer to the
percentage of sequence similarity found in a comparison of two or
more amino acid or nucleic acid sequences. Percent identity can be
determined electronically, e.g., by using the MegAlign.TM. program
(DNASTAR, Inc., Madison Wis.). The MegAlign.TM. program can create
alignments between two or more sequences according to different
methods, e.g., the clustal method. (See, e.g., Higgins, D. G. and
P. M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groups
sequences into clusters by examining the distances between all
pairs. The clusters are aligned pairwise and then in groups. The
percentage similarity between two amino acid sequences, e.g.,
sequence A and sequence B, is calculated by dividing the length of
sequence A, minus the number of gap residues in sequence A, minus
the number of gap residues in sequence B, into the sum of the
residue matches between sequence A and sequence B, times one
hundred. Gaps of low or of no homology between the two amino acid
sequences are not included in determining percentage similarity.
Percent identity between nucleic acid sequences can also be counted
or calculated by other methods known in the art, e.g., the Jotun
Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol.
183:626-645.) Identity between sequences can also be determined by
other methods known in the art, e.g., by varying hybridization
conditions.
[0048] "Human artificial chromosomes" (HACs), as described herein,
are linear microchromosomes which may contain DNA sequences of
about 6 kb to 10 Mb in size, and which contain all of the elements
required for stable mitotic chromosome segregation and maintenance.
(See, e.g., Harrington, J. J. et al. (1997) Nat Genet.
15:345-355.)
[0049] The term "humanized antibody," as used herein, refers to
antibody molecules in which the amino acid sequence in the
non-antigen binding regions has been altered so that the antibody
more closely resembles a human antibody, and still retains its
original binding ability.
[0050] "Hybridization," as the term is used herein, refers to any
process by which a strand of nucleic acid binds with a
complementary strand through base pairing.
[0051] As used herein, the term "hybridization complex" as used
herein, refers to a complex formed between two nucleic acid
sequences by virtue of the formation of hydrogen bonds between
complementary bases. A hybridization complex may be formed in
solution (e.g., C.sub.0t or R.sub.0t analysis) or formed between
one nucleic acid sequence present in solution and another nucleic
acid sequence immobilized on a solid support (e.g., paper,
membranes, filters, chips, pins or glass slides, or any other
appropriate substrate to which cells or their nucleic acids have
been fixed).
[0052] The words "insertion" or "addition," as used herein, refer
to changes in an amino acid or nucleotide sequence resulting in the
addition of one or more amino acid residues or nucleotides,
respectively, to the sequence found in the naturally occurring
molecule.
[0053] "Immune response" can refer to conditions associated with
immunological disorders, trauma, immune disorders, or infectious or
genetic disease, etc. These conditions can be characterized by
expression of various factors, e.g., cytokines, chemokines, and
other signaling molecules, which may affect cellular and systermic
defense systems.
[0054] The term "microarray," as used herein, refers to an
arrangement of distinct polynucleotides arrayed on a substrate,
e.g., paper, nylon or any other type of membrane, filter, chip,
glass slide, or any other suitable solid support.
[0055] The terms "element" or "array element" as used herein in a
microarray context, refer to hybridizable polynucleotides arranged
on the surface of a substrate.
[0056] The term "modulate," as it appears herein, refers to a
change in the activity of IRS-p53h. For example, modulation may
cause an increase or a decrease in protein activity, binding
characteristics, or any other biological, functional, or
immunological properties of IRS-p53h.
[0057] The phrases "nucleic acid" or "nucleic acid sequence," as
used herein, refer to an oligonucleotide, nucleotide,
polynucleotide, or any fragment thereof, to DNA or RNA of genomic
or synthetic origin which may be single-stranded or double-stranded
and may represent the sense or the antisense strand, to peptide
nucleic acid (PNA), or to any DNA-like or RNA-like material. In
this context, "fragments" refers to those nucleic acid sequences
which are greater than about 60 nucleotides in length, and most
preferably are at least about 100 nucleotides, at least about 1000
nucleotides, or at least about 10,000 nucleotides in length.
[0058] The terms "operably associated" or "operably linked," as
used herein, refer to functionally related nucleic acid sequences.
A promoter is operably associated or operably linked with a coding
sequence if the promoter controls the transcription of the encoded
polypeptide. While operably associated or operably linked nucleic
acid sequences can be contiguous and in the same reading frame,
certain genetic elements, e.g., repressor genes, are not
contiguously linked to the sequence encoding the polypeptide but
still bind to operator sequences that control expression of the
polypeptide.
[0059] The term "oligonucleotide," as used herein, refers to a
nucleic acid sequence of at least about 6 nucleotides to 60
nucleotides, preferably about 15 to 30 nucleotides, and most
preferably about 20 to 25 nucleotides, which can be used in PCR
amplification or in a hybridization assay or microarray. As used
herein, the term "oligonucleotide" is substantially equivalent to
the terms "amplimer," "primer," "oligomer," and "probe," as these
terms are commonly defined in the art.
[0060] "Peptide nucleic acid" (PNA), as used herein, refers to an
antisense molecule or anti-gene agent which comprises an
oligonucleotide of at least about 5 nucleotides in length linked to
a peptide backbone of amino acid residues ending in lysine. The
terminal lysine confers solubility to the composition. PNAs
preferentially bind complementary single stranded DNA and RNA and
stop transcript elongation, and may be pegylated to extend their
lifespan in the cell. (See, e.g., Nielsen, P. E. et al. (1993)
Anticancer Drug Des. 8:53-63.)
[0061] The term "sample," as used herein, is used in its broadest
sense. A biological sample suspected of containing nucleic acids
encoding IRS-p53h, or fragments thereof, or IRS-p53h itself, may
comprise a bodily fluid; an extract from a cell, chromosome,
organelle, or membrane isolated from a cell; a cell; genomic DNA,
RNA, or cDNA, in solution or bound to a solid support; a tissue; a
tissue print; etc.
[0062] As used herein, the terms "specific binding" or
"specifically binding" refer to that interaction between a protein
or peptide and an agonist, an antibody, or an antagonist. The
interaction is dependent upon the presence of a particular
structure of the protein, e.g., the antigenic determinant or
epitope, recognized by the binding molecule. For example, if an
antibody is specific for epitope "A," the presence of a polypeptide
containing the epitope A, or the presence of free unlabeled A, in a
reaction containing free labeled A and the antibody will reduce the
amount of labeled A that binds to the antibody.
[0063] As used herein, the term "stringent conditions" refers to
conditions which permit hybridization between polynucleotide
sequences and the claimed polynucleotide sequences. Suitably
stringent conditions can be defined by, for example, the
concentrations of salt or formamide in the prehybridization and
hybridization solutions, or by the hybridization temperature, and
are well known in the art. In particular, stringency can be
increased by reducing the concentration of salt, increasing the
concentration of formamide, or raising the hybridization
temperature.
[0064] For example, hybridization under high stringency conditions
could occur in about 50% formamide at about 37.degree. C. to
42.degree. C. Hybridization could occur under reduced stringency
conditions in about 35% to 25% formamide at about 30.degree. C. to
35.degree. C. In particular, hybridization could occur under high
stringency conditions at 42.degree. C. in 50% formamide, 5.times.
SSPE, 0.3% SDS, and 200 .mu.g/ml sheared and denatured salmon sperm
DNA. Hybridization could occur under reduced stringency conditions
as described above, but in 35% formamide at a reduced temperature
of 35.degree. C. The temperature range corresponding to a
particular level of stringency can be further narrowed by
calculating the purine to pyrimidine ratio of the nucleic acid of
interest and adjusting the temperature accordingly. Variations on
the above ranges and conditions are well known in the art.
[0065] The term "substantially purified," as used herein, refers to
nucleic acid or amino acid sequences that are removed from their
natural environment and are isolated or separated, and are at least
about 60% free, preferably about 75% free, and most preferably
about 90% free from other components with which they are naturally
associated.
[0066] A "substitution," as used herein, refers to the replacement
of one or more amino acids or nucleotides by different amino acids
or nucleotides, respectively.
[0067] "Transformation," as defined herein, describes a process by
which exogenous DNA enters and changes a recipient cell.
Transformation may occur under natural or artificial conditions
according to various methods well known in the art, and may rely on
any known method for the insertion of foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method
for transformation is selected based on the type of host cell being
transformed and may include, but is not limited to, viral
infection, electroporation, heat shock, lipofection, and particle
bombardment. The term "transformed" cells includes stably
transformed cells in which the inserted DNA is capable of
replication either as an autonomously replicating plasmid or as
part of the host chromosome, as well as transiently transformed
cells which express the inserted DNA or RNA for limited periods of
time.
[0068] A "variant" of IRS-p53h, as used herein, refers to an amino
acid sequence that is altered by one or more amino acids. The
variant may have "conservative" changes, wherein a substituted
amino acid has similar structural or chemical properties (e.g.,
replacement of leucine with isoleucine). More rarely, a variant may
have "nonconservative" changes (e.g., replacement of glycine with
tryptophan). Analogous minor variations may also include amino acid
deletions or insertions, or both. Guidance in determining which
amino acid residues may be substituted, inserted, or deleted
without abolishing biological or immunological activity may be
found using computer programs well known in the art, for example,
LASERGENE.TM. software.
THE INVENTION
[0069] The invention is based on the discovery of two new human
insulin receptor tyrosine kinase substrates (collectively referred
to as IRS-p53h, and individually as IRS-p53h-1 and IRS-p53h-2), the
polynucleotides encoding IRS-p53h, and the use of these
compositions for the diagnosis, treatment, or prevention of
reproductive disorders, Alzheimer's disease, cancer, immunological
disorders, and disorders associated with insulin response.
[0070] Nucleic acids encoding the IRS-p53h-1 of the present
invention were first identified in Incyte Clone 918158 from a
carcinoma-associated breast tissue cDNA library (BRSTNOT04) using a
computer search for amino acid sequence alignments. A consensus
sequence, SEQ ID NO:2, was derived from the following overlapping
and/or extended nucleic acid sequences: Incyte Clones 918158
(BRSTNOT04), 1342719 (COLNTUT03), and 1522281 (BLADTUT04).
[0071] Nucleic acids encoding the IRS-p53h-2 of the present
invention were first identified in Incyte Clone 2493150 from the
adrenal cDNA library (ADRETUT05) using a computer search for amino
acid sequence alignments. A consensus sequence, SEQ ID NO:4, was
derived from the following overlapping and/or extended nucleic acid
sequences: Incyte Clones 2493150 (ADRETUT05), 918158 (BRSTNOT04),
1520586 and 1522281 (BLADTUT04), 1989307 (CORPNOT02), 1263953
(SYNORAT05), and 1466687 (PANCTUT02).
[0072] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:1, as shown in
FIGS. 1B, 1C, 1D, 1E, and 1F. IRS-p53h-1 is 534 amino acids in
length and contains two potential N-glycosylation sites at residues
N358 and N390; one potential cAMP- and cGMP-dependent protein
kinase phosphorylation site at residue S148; eight potential casein
kinase II phosphorylation sites at residues S4, T19, T103, S129,
S158, S291, T303, and S395; seven potential protein kinase C
phosphorylation sites at residues S27, S158, S169, T348, S395,
S418, and S440; and three potential tyrosine kinase phosphorylation
sites at residues Y17, Y115, and Y178. As shown in FIGS. 3A, 3B,
3C, and 3D, IRS-p53h-1 has chemical and structural homology with
IRS p53 from hamster (GI 1203820; SEQ ID NO:5). In particular,
IRS-p53h-1 and hamster IRS p53 share 93% amino acid sequence
identity, the two potential N-glycosylation sites, the potential
cAMP- and cGMP-dependent protein kinase phosphorylation site, seven
potential casein kinase II phosphorylation sites, seven potential
protein kinase C phosphorylation sites, and three potential
tyrosine kinase phosphorylation sites. A fragment of SEQ ID NO:2
from about nucleotide 1621 to about nucleotide 1647 is useful for
designing oligonucleotides or to be used directly as a
hybridization probe. Northern analysis shows the expression of
IRS-p53h-1 in various tissues, at least 57% of which are
immortalized or cancerous and at least 19% of which involve immune
response. Of particular note is the expression of IRS-p53h-1 in
reproductive, brain, and gastrointestinal tissues, and in
Alzheimer's disease.
[0073] In another embodiment, the invention encompasses a
polypeptide comprising the amino acid sequence of SEQ ID NO:3, as
shown in FIGS. 2A, 2B, 2C, 2D, 2E, and 2F. IRS-p53h-2 is 553 amino
acids in length and has two potential N-glycosylation sites at
residues N391 and N423; one potential cAMP- and cGMP-dependant
protein kinase phosphorylation site at residue S181; nine potential
casein kinase II phosphorylation sites at residues S4, T52, T136,
S162, S191, S324, T336, S428, and S544; seven potential protein
kinase C phosphorylation sites at residues S60, S191, S202, T381,
S428, S451, and S473; four potential tyrosine kinase
phosphorylation sites at residues Y17, Y24, Y148, and Y211; and a
microbodies C-terminal targeting signal from about residue A551 to
about residue F553. As shown in FIGS. 3A, 3B, 3C, and 3D,
IRS-p53h-2 has chemical and structural homology with IRS-p53h-1,
and are considered to be splice variants. In addition, as shown in
FIGS. 3A, 3B, 3C, and 3D, IRS-pS3h-2 has chemical and structural
homology with IRS-p53 from hamster (GI 1203820; SEQ ID NO:5). In
particular, IRS-p53h-2 and IRS-p53 from hamster share 90% identity,
the two potential N-glycosylation sites, the potential cAMP- and
cGMP-dependent protein kinase phosphorylation site, seven potential
casein kinase II phosphorylation sites, seven potential protein
kinase C phosphorylation sites, three potential tyrosine kinase
phosphorylation sites; and the microbodies C-terminal targeting
signal. A fragment of SEQ ID NO:4 from about nucleotide 295 to
about nucleotide 315 is useful for designing oligonucleotides or to
be used directly as a hybridization probe. Northern analysis shows
the expression of this sequence in various libraries, at least 61%
of which are immortalized or cancerous and at least 14% of which
involve immune response. Of particular note is the expression of
IRS-p53h-2 in reproductive, brain, gastrointestinal, lung tissues,
and in Alzheimer's disease.
[0074] The invention also encompasses IRS-p53h variants. A
preferred IRS-p53h variant is one which has at least about 80%,
more preferably at least about 90%, and most preferably at least
about 95% amino acid sequence identity to the IRS-p53h amino acid
sequence, and which contains at least one functional or structural
characteristic of IRS-p53h.
[0075] The invention also encompasses polynucleotides which encode
IRS-p53h. In a particular embodiment, the invention encompasses a
polynucleotide sequence comprising the sequence of SEQ ID NO:2,
which encodes an IRS-p53h, as shown in FIGS. 1A, 1B, 1C, 1D, 1E,
and 1F. In a further embodiment, the invention encompasses the
polynucleotide sequence comprising the sequence of SEQ ID NO:4, as
shown in FIGS. 2A, 2B, 2C, 2D, 2E, and 2F.
[0076] The invention also encompasses a variant of a polynucleotide
sequence encoding IRS-p53h. In particular, such a variant
polynucleotide sequence will have at least about 80%, more
preferably at least about 90%, and most preferably at least about
95% polynucleotide sequence identity to the polynucleotide sequence
encoding IRS-p53h. A particular aspect of the invention encompasses
a variant of SEQ ID NO:2 which has at least about 80%, more
preferably at least about 90%, and most preferably at least about
95% polynucleotide sequence identity to SEQ ID NO:2. The invention
further encompasses a polynucleotide variant of SEQ ID NO:4 having
at least about 80%, more preferably at least about 90%, and most
preferably at least about 95% polynucleotide sequence identity to
SEQ ID NO:4. Any one of the polynucleotide variants described above
can encode an amino acid sequence which contains at least one
functional or structural characteristic of IRS-p53h.
[0077] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
polynucleotide sequences encoding IRS-p53h, some bearing minimal
homology to the polynucleotide sequences of any known and naturally
occurring gene, may be produced. Thus, the invention contemplates
each and every possible variation of polynucleotide sequence that
could be made by selecting combinations based on possible codon
choices. These combinations are made in accordance with the
standard triplet genetic code as applied to the polynucleotide
sequence of naturally occurring IRS-p53h, and all such variations
are to be considered as being specifically disclosed.
[0078] Although nucleotide sequences which encode IRS-p53h and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring IRS-p53h under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding IRS-p53h or its derivatives
possessing a substantially different codon usage. Codons may be
selected to increase the rate at which expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance
with the frequency with which particular codons are utilized by the
host. Other reasons for substantially altering the nucleotide
sequence encoding IRS-p53h and its derivatives without altering the
encoded amino acid sequences include the production of RNA
transcripts having more desirable properties, such as a greater
half-life, than transcripts produced from the naturally occurring
sequence.
[0079] The invention also encompasses production of DNA sequences
which encode IRS-p53h and IRS-p53h derivatives, or fragments
thereof, entirely by synthetic chemistry. After production, the
synthetic sequence may be inserted into any of the many available
expression vectors and cell systems using reagents that are well
known in the art. Moreover, synthetic chemistry may be used to
introduce mutations into a sequence encoding IRS-p53h or any
fragment thereof.
[0080] Also encompassed by the invention are polynucleotide
sequences that are capable of hybridizing to the claimed
polynucleotide sequences, and, in particular, to those shown in SEQ
ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, or a fragment of
SEQ ID NO:4, under various conditions of stringency. (See, e.g.,
Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399-407;
Kimmel, A. R. (1987) Methods Enzymol. 152:507-511.)
[0081] Methods for DNA sequencing are well known and generally
available in the art and may be used to practice any of the
embodiments of the invention. The methods may employ such enzymes
as the Kienow fragment of DNA polymerase I, Sequenase.RTM. (US
Biochemical Corp., Cleveland, Ohio), Taq polymerase (Perkin Elmer),
thermostable T7 polymerase (Amersham, Chicago, Ill.), or
combinations of polymerases and proofreading exonucleases such as
those found in the ELONGASE Amplification System (GIBCO/BRL,
Gaithersburg, Md.). Preferably, the process is automated with
machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno,
Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,
Mass.) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin
Elmer).
[0082] The nucleic acid sequences encoding IRS-p53h may be extended
utilizing a partial nucleotide sequence and employing various
methods known in the art to detect upstream sequences, such as
promoters and regulatory elements. For example, one method which
may be employed, restriction-site PCR, uses universal primers to
retrieve unknown sequence adjacent to a known locus. (See, e.g.,
Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) In particular,
genomic DNA is first amplified in the presence of a primer which is
complementary to a linker sequence within the vector and a primer
specific to a region of the nucleotide sequence. The amplified
sequences are then subjected to a second round of PCR with the same
linker primer and another specific primer internal to the first
one. Products of each round of PCR are transcribed with an
appropriate RNA polymerase and sequenced using reverse
transcriptase.
[0083] Inverse PCR may also be used to amplify or extend sequences
using divergent primers based on a known region. (See, e.g.,
Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) The primers
may be designed using commercially available software such as OLIGO
4.06 Primer Analysis software (National Biosciences Inc., Plymouth,
Minn.) or another appropriate program to be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more,
and to anneal to the target sequence at temperatures of about
68.degree. C. to 72.degree. C. The method uses several restriction
enzymes to generate a suitable fragment in the known region of a
gene. The fragment is then circularized by intramolecular ligation
and used as a PCR template.
[0084] Another method which may be used is capture PCR, which
involves PCR amplification of DNA fragments adjacent to a known
sequence in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119.) In
this method, multiple restriction enzyme digestions and ligations
may be used to place an engineered double-stranded sequence into an
unknown fragment of the DNA molecule before performing PCR. Other
methods which may be used to retrieve unknown sequences are known
in the art. (See, e.g., Parker, J. D. et al. (1991) Nucleic Acids
Res. 19:3055-3060.) Additionally, one may use PCR, nested primers,
and PromoterFinder.TM. libraries to walk genomic DNA (Clontech,
Palo Alto, Calif.). This process avoids the need to screen
libraries and is useful in finding intron/exon junctions.
[0085] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
Also, random-primed libraries are preferable in that they will
include more sequences which contain the 5' regions of genes. Use
of a randomly primed library may be especially preferable for
situations in which an oligo d(T) library does not yield a
full-length cDNA. Genomic libraries may be useful for extension of
sequence into non-transcribed regulatory regions.
[0086] Capillary electrophoresis systems which are commercially
available may be used to analyze the size or confirm the nucleotide
sequence of sequencing or PCR products. In particular, capillary
sequencing may employ flowable polymers for electrophoretic
separation, four different fluorescent dyes (one for each
nucleotide) which are laser activated, and a charge coupled device
camera for detection of the emitted wavelengths. Output/light
intensity may be converted to electrical signal using appropriate
software (e.g., Genotyper.TM. and Sequence Navigator.TM., Perkin
Elmer), and the entire process from loading of samples to computer
analysis and electronic data display may be computer controlled.
Capillary electrophoresis is especially preferable for the
sequencing of small pieces of DNA which might be present in limited
amounts in a particular sample.
[0087] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode IRS-p53h may be used in
recombinant DNA molecules to direct expression of IRS-p53h, or
fragments or functional equivalents thereof, in appropriate host
cells. Due to the inherent degeneracy of the genetic code, other
DNA sequences which encode substantially the same or a functionally
equivalent amino acid sequence may be produced, and these sequences
may be used to clone and express IRS-p53h.
[0088] As will be understood by those of skill in the art, it may
be advantageous to produce IRS-p53h-encoding nucleotide sequences
possessing non-naturally occurring codons. For example, codons
preferred by a particular prokaryotic or eukaryotic host can be
selected to increase the rate of protein expression or to produce
an RNA transcript having desirable properties, such as a half-life
which is longer than that of a transcript generated from the
naturally occurring sequence.
[0089] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter IRS-p53h-encoding sequences for a variety of reasons
including, but not limited to, alterations which modify the
cloning, processing, and/or expression of the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene
fragments and synthetic oligonucleotides may be used to engineer
the nucleotide sequences. For example, site-directed mutagenesis
may be used to insert new restriction sites, alter glycosylation
patterns, change codon preference, produce splice variants,
introduce mutations, and so forth.
[0090] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding IRS-p53h may be
ligated to a heterologous sequence to encode a fusion protein. For
example, to screen peptide libraries for inhibitors of IRS-p53h
activity, it may be useful to encode a chimeric IRS-p53h protein
that can be recognized by a commercially available antibody. A
fusion protein may also be engineered to contain a cleavage site
located between the IRS-p53h encoding sequence and the heterologous
protein sequence, so that IRS-p53h may be cleaved and purified away
from the heterologous moiety.
[0091] In another embodiment, sequences encoding IRS-p53h may be
synthesized, in whole or in part, using chemical methods well known
in the art. (See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids
Res. Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids
Res. Symp. Ser. 225-232.) Alternatively, the protein itself may be
produced using chemical methods to synthesize the amino acid
sequence of IRS-p53h, or a fragment thereof. For example, peptide
synthesis can be performed using various solid-phase techniques.
(See, e.g., Roberge, J. Y. et al. (1995) Science 269:202-204.)
Automated synthesis may be achieved using the ABI 431A Peptide
Synthesizer (Perkin Elmer). Additionally, the amino acid sequence
of IRS-p53h, or any part thereof, may be altered during direct
synthesis and/or combined with sequences from other proteins, or
any part thereof, to produce a variant polypeptide.
[0092] The peptide may be substantially purified by preparative
high performance liquid chromatography. (See, e.g, Chiez, R. M. and
F. Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition
of the synthetic peptides may be confirmed by amino acid analysis
or by sequencing. (See, e.g., Creighton, T. (1984) Proteins,
Structures and Molecular Properties, W H Freeman and Co., New York,
N.Y.)
[0093] In order to express a biologically active IRS-p53h, the
nucleotide sequences encoding IRS-p53h or derivatives thereof may
be inserted into appropriate expression vector, i.e., a vector
which contains the necessary elements for the transcription and
translation of the inserted coding sequence.
[0094] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding IRS-p53h and appropriate transcriptional and translational
control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview,
N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al. (1995, and
periodic supplements) Current Protocols in Molecular Biology, John
Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)
[0095] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding IRS-p53h. These include,
but are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with virus expression vectors (e.g.,
baculovirus); plant cell systems transformed with virus expression
vectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic
virus (TMV)) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems. The invention is not
limited by the host cell employed.
[0096] The "control elements" or "regulatory sequences" are those
non-translated regions, e.g., enhancers, promoters, and 5' and 3'
untranslated regions, of the vector and polynucleotide sequences
encoding IRS-p53h which interact with host cellular proteins to
carry out transcription and translation. Such elements may vary in
their strength and specificity. Depending on the vector system and
host utilized, any number of suitable transcription and translation
elements, including constitutive and inducible promoters, may be
used. For example, when cloning in bacterial systems, inducible
promoters, e.g., hybrid lacZ promoter of the Bluescript.RTM.
phagemid (Stratagene, La Jolla, Calif.) or pSport.TM. plasmid
(GIBCO/BRL), may be used. The baculovirus polyhedrin promoter may
be used in insect cells. Promoters or enhancers derived from the
genomes of plant cells (e.g., heat shock, RUBISCO, and storage
protein genes) or from plant viruses (e.g., viral promoters or
leader sequences) may be cloned into the vector. In mammalian cell
systems, promoters from mammalian genes or from mammalian viruses
are preferable. If it is necessary to generate a cell line that
contains multiple copies of the sequence encoding IRS-p53h, vectors
based on SV40 or EBV may be used with an appropriate selectable
marker.
[0097] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for IRS-p53h. For example,
when large quantities of IRS-p53h are needed for the induction of
antibodies, vectors which direct high level expression of fusion
proteins that are readily purified may be used. Such vectors
include, but are not limited to, multifunctional E. coli cloning
and expression vectors such as Bluescript.RTM. (Stratagene), in
which the sequence encoding IRS-p53h may be ligated into the vector
in frame with sequences for the amino-terminal Met and the
subsequent 7 residues of .beta.-galactosidase so that a hybrid
protein is produced, and pIN vectors. (See, e.g., Van Heeke, G. and
S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) pGEX vectors
(Amersham Pharmacia Biotech, Uppsala, Sweden) may also be used to
express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption to
glutathione-agarose beads followed by elution in the presence of
free glutathione. Proteins made in such systems may be designed to
include heparin, thrombin, or factor XA protease cleavage sites so
that the cloned polypeptide of interest can be released from the
GST moiety at will.
[0098] In the yeast Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters, such as alpha
factor, alcohol oxidase, and PGH, may be used. (See, e.g., Ausubel,
supra; and Grant et al. (1987) Methods Enzymol. 153:516-544.)
[0099] In cases where plant expression vectors are used, the
expression of sequences encoding IRS-p53h may be driven by any of a
number of promoters. For example, viral promoters such as the 35S
and 19S promoters of CaMV may be used alone or in combination with
the omega leader sequence from TMV. (Takamatsu, N. (1987) EMBO J.
6:307-311.) Alternatively, plant promoters such as the small
subunit of RUBISCO or heat shock promoters may be used. (See, e.g.,
Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results
Probl. Cell Differ. 17:85-105.) These constructs can be introduced
into plant cells by direct DNA transformation or pathogen-mediated
transfection. Such techniques are described in a number of
generally available reviews. (See, e.g., Hobbs, S. or Murry, L.E.
in McGraw Hill Yearbook of Science and Technology (1992) McGraw
Hill, New York, N.Y.; pp. 191-196.)
[0100] An insect system may also be used to express IRS-p53h. For
example, in one such system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The
sequences encoding IRS-p53h may be cloned into a non-essential
region of the virus, such as the polyhedrin gene, and placed under
control of the polyhedrin promoter. Successful insertion of
sequences encoding IRS-p53h will render the polyhedrin gene
inactive and produce recombinant virus lacking coat protein. The
recombinant viruses may then be used to infect, for example, S.
frugiperda cells or Trichoplusia larvae in which IRS-p53h may be
expressed. (See, e.g., Engelhard, E. K. et al. (1994) Proc. Nat.
Acad. Sci. 91:3224-3227.)
[0101] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding IRS-p53h may be ligated into
an adenovirus transcription/translation complex consisting of the
late promoter and tripartite leader sequence. Insertion in a
non-essential E1 or E3 region of the viral genome may be used to
obtain a viable virus which is capable of expressing IRS-p53h in
infected host cells. (See, e.g., Logan, J. and T. Shenk (1984)
Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcription
enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be
used to increase expression in mammalian host cells.
[0102] Human artificial chromosomes (HACs) may also be employed to
deliver larger fragments of DNA than can be contained and expressed
in a plasmid. HACs of about 6 kb to 10 Mb are constructed and
delivered via conventional delivery methods (liposomes,
polycationic amino polymers, or vesicles) for therapeutic
purposes.
[0103] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding IRS-p53h. Such signals
include the ATG initiation codon and adjacent sequences. In cases
where sequences encoding IRS-p53h and its initiation codon and
upstream sequences are inserted into the appropriate expression
vector, no additional transcriptional or translational control
signals may be needed. However, in cases where only coding
sequence, or a fragment thereof, is inserted, exogenous
translational control signals including the ATG initiation codon
should be provided. Furthermore, the initiation codon should be in
the correct reading frame to ensure translation of the entire
insert. Exogenous translational elements and initiation codons may
be of various origins, both natural and synthetic. The efficiency
of expression may be enhanced by the inclusion of enhancers
appropriate for the particular cell system used. (See, e.g.,
Scharf, D. et al. (1994) Results Probl. Cell Differ.
20:125-162.)
[0104] In addition, a host cell strain may be chosen for its
ability to modulate expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to
facilitate correct insertion, folding, and/or function. Different
host cells which have specific cellular machinery and
characteristic mechanisms for post-translational activities (e.g.,
CHO, HeLa, MDCK, HEK293, and WI38), are available from the American
Type Culture Collection (ATCC, Bethesda, Md.) and may be chosen to
ensure the correct modification and processing of the foreign
protein.
[0105] For long term, high yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
capable of stably expressing IRS-p53h can be transformed using
expression vectors which may contain viral origins of replication
and/or endogenous expression elements and a selectable marker gene
on the same or on a separate vector. Following the introduction of
the vector, cells may be allowed to grow for about 1 to 2 days in
enriched media before being switched to selective media. The
purpose of the selectable marker is to confer resistance to
selection, and its presence allows growth and recovery of cells
which successfully express the introduced sequences. Resistant
clones of stably transformed cells may be proliferated using tissue
culture techniques appropriate to the cell type.
[0106] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase genes and adenine
phosphoribosyltransferase genes, which can be employed in tk.sup.-
or apr.sup.- cells, respectively. (See, e.g., Wigler, M. et al.
(1977) Cell 11:223-232; and Lowy, I. et al. (1980) Cell
22:817-823.) Also, antimetabolite, antibiotic, or herbicide
resistance can be used as the basis for selection. For example,
dhfr confers resistance to methotrexate; npt confers resistance to
the aminoglycosides neomycin and G-418; and als or pat confer
resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl.
Acad. Sci. 77:3567-3570; Colbere-Garapin, F. et al (1981) J. Mol.
Biol. 150:1-14; and Murry, supra.) Additional selectable genes have
been described, e.g., trpB, which allows cells to utilize indole in
place of tryptophan, or hisd, which allows cells to utilize
histinol in place of histidine. (See, e.g., Hartman, S. C. and R.
C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-8051.) Visible
markers, e.g., anthocyanins, .beta. glucuronidase and its substrate
GUS, luciferase and its substrate luciferin may be used. Green
fluorescent proteins (GFP) (Clontech, Palo Alto, Calif.) can also
be used. These markers can be used not only to identify
transformants, but also to quantify the amount of transient or
stable protein expression attributable to a specific vector system.
(See, e.g., Rhodes, C. A. et al. (1995) Methods Mol. Biol.
55:121-131.)
[0107] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, the presence
and expression of the gene may need to be confirmed. For example,
if the sequence encoding IRS-p53h is inserted within a marker gene
sequence, transformed cells containing sequences encoding IRS-p53h
can be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding IRS-p53h under the control of a single promoter.
Expression of the marker gene in response to induction or selection
usually indicates expression of the tandem gene as well.
[0108] Alternatively, host cells which contain the nucleic acid
sequence encoding IRS-p53h and express IRS-p53h may be identified
by a variety of procedures known to those of skill in the art.
These procedures include, but are not limited to, DNA-DNA or
DNA-RNA hybridizations and protein bioassay or immunoassay
techniques which include membrane, solution, or chip based
technologies for the detection and/or quantification of nucleic
acid or protein sequences.
[0109] The presence of polynucleotide sequences encoding IRS-p53h
can be detected by DNA-DNA or DNA-RNA hybridization or
amplification using probes or fragments or fragments of
polynucleotides encoding IRS-p53h. Nucleic acid amplification based
assays involve the use of oligonucleotides or oligomers based on
the sequences encoding IRS-p53h to detect transformants containing
DNA or RNA encoding IRS-p53h.
[0110] A variety of protocols for detecting and measuring the
expression of IRS-p53h, using either polyclonal or monoclonal
antibodies specific for the protein, are known in the art. Examples
of such techniques include enzyme-linked immunosorbent assays
(ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell
sorting (FACS). A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on
IRS-p53h is preferred, but a competitive binding assay may be
employed. These and other assays are well described in the art.
(See, e.g., Hampton, R. et al. (1990) Serological Methods, a
Laboratory Manual, APS Press, St Paul, Minn., Section IV; and
Maddox, D. E. et al. (1983) J. Exp. Med. 158:1211-1216).
[0111] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides encoding IRS-p53h include oligolabeling, nick
translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding IRS-p53h, or any
fragments thereof, may be cloned into a vector for the production
of an mRNA probe. Such vectors are known in the art, are
commercially available, and may be used to synthesize RNA probes in
vitro by addition of an appropriate RNA polymerase such as T7, T3,
or SP6 and labeled nucleotides. These procedures may be conducted
using a variety of commercially available kits, such as those
provided by Pharmacia & Upjohn (Kalamazoo, Mich.), Promega
(Madison, Wis.), and U.S. Biochemical Corp. (Cleveland, Ohio).
Suitable reporter molecules or labels which may be used for ease of
detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic agents, as well as substrates,
cofactors, inhibitors, magnetic particles, and the like.
[0112] Host cells transformed with nucleotide sequences encoding
IRS-p53h may be cultured under conditions suitable for the
expression and recovery of the protein from cell culture. The
protein produced by a transformed cell may be secreted or contained
intracellularly depending on the sequence and/or the vector used.
As will be understood by those of skill in the art, expression
vectors containing polynucleotides which encode IRS-p53h may be
designed to contain signal sequences which direct secretion of
IRS-p53h through a prokaryotic or eukaryotic cell membrane. Other
constructions may be used to join sequences encoding IRS-p53h to
nucleotide sequences encoding a polypeptide domain which will
facilitate purification of soluble proteins. Such purification
facilitating domains include, but are not limited to, metal
chelating peptides such as histidine-tryptophan modules that allow
purification on immobilized metals, protein A domains that allow
purification on immobilized immunoglobulin, and the domain utilized
in the FLAGS extension/affinity purification system (Immunex Corp.,
Seattle, Wash.). The inclusion of cleavable linker sequences, such
as those specific for Factor XA or enterokinase (Invitrogen, San
Diego, Calif.), between the purification domain and the IRS-p53h
encoding sequence may be used to facilitate purification. One such
expression vector provides for expression of a fusion protein
containing IRS-p53h and a nucleic acid encoding 6 histidine
residues preceding a thioredoxin or an enterokinase cleavage site.
The histidine residues facilitate purification on immobilized metal
ion affinity chromatography (IMAC). (See, e.g., Porath, J. et al.
(1992) Prot. Exp. Purif. 3: 263-281.) The enterokinase cleavage
site provides a means for purifying IRS-p53h from the fusion
protein. (See, e.g., Kroll, D. J. et al. (1993) DNA Cell Biol.
12:441-453.)
[0113] Fragments of IRS-p53h may be produced not only by
recombinant production, but also by direct peptide synthesis using
solid-phase techniques. (See, e.g., Creighton, supra pp. 55-60.)
Protein synthesis may be performed by manual techniques or by
automation. Automated synthesis may be achieved, for example, using
the Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer).
Various fragments of IRS-p53h may be synthesized separately and
then combined to produce the full length molecule.
THERAPEUTICS
[0114] Chemical and structural homology exits between IRS-p53h and
IRS p53 from hamster (GI 1203820). In addition, IRS-p53h is
expressed in reproductive, brain, gastrointestinal, and lung
tissues, and in Alzheimer's disease. Therefore, IRS-p53h appears to
play a role in reproductive disorders, Alzheimer's disease, cancer,
immunological disorders, and disorders associated with insulin
response. In particular, increased expression or activity of
IRS-p53h appears to be associated with cancer or immunological
disorders, and decreased expression or activity of IRS-p53h with a
role in disorders associated with insulin response.
[0115] Therefore, in one embodiment, IRS-p53h or a fragment or
derivative thereof may be administered to a subject to treat a
disorder associated with insulin response. Such a disorder may
include, but is not limited to, type 2 (non-insulin-dependent)
diabetes mellitus, hyperglycemia, myotonic muscular dystrophy,
acanthosis nigricans, retinopathy, nephropathy, atherosclerotic
coronary and peripheral arterial disease, and peripheral and
autonomic neuropathies.
[0116] In another embodiment, a vector capable of expressing
IRS-p53h, or a fragment or a derivative thereof, may also be
administered to a subject to treat a disorder associated with
insulin response, including but not limited to those listed
above.
[0117] In a further embodiment, a pharmaceutical composition
comprising a substantially purified IRS-p53h in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a reproductive disorder including, but not limited
to, those provided above.
[0118] In still another embodiment, an agonist of IRS-p53h may also
be administered to a subject to treat a disorder associated with
insulin response, including but not limited to those listed
above.
[0119] In another embodiment, an antagonist of IRS-p53h may be
administered to a subject to treat or prevent a reproductive
disorder. Such a reproductive disorder may include, but is not
limited to, disorders of prolactin production; infertility,
including tubal disease, ovulatory defects, and endometriosis;
disruptions of the estrous cycle, disruptions of the menstrual
cycle, polycystic ovary syndrome, ovarian hyperstimulation
syndrome, endometrial and ovarian tumors, autoimmune disorders,
ectopic pregnancy, and teratogenesis; cancer of the breast, uterine
fibroids, fibrocystic breast disease, galactorrhea; disruptions of
spermatogenesis, abnormal sperm physiology, cancer of the testis,
cancer of the prostate, benign prostatic hyperplasia, prostatitis,
Peyronie's disease, carcinoma of the male breast and gynecomastia.
In one aspect, an antibody which specifically binds IRS-p53h may be
used directly as an antagonist or indirectly as a targeting or
delivery mechanism for bringing a pharmaceutical agent to cells or
tissue which express IRS-p53h.
[0120] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding IRS-p53h may be
administered to a subject to treat or prevent a reproductive
disorder including, but not limited to, those described above.
[0121] In another embodiment, an antagonist of IRS-p53h may be
administered to a subject to treat or prevent Alzheimer's disease.
In one aspect, an antibody which specifically binds IRS-p53h may be
used directly as an antagonist or indirectly as a targeting or
delivery mechanism for bringing a pharmaceutical agent to cells or
tissue which express IRS-p53h.
[0122] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding IRS-p53h may be
administered to a subject to treat or prevent Alzheimer's
disease.
[0123] In another embodiment, an antagonist of IRS-p53h may be
administered to a subject to prevent or treat cancer. Such cancers
include, but are not limited to, adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, and
particularly cancers of the adrenal gland, bladder, bone, bone
marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus. In one aspect,
antibodies which specifically bind IRS-p53h may be used directly as
an antagonist or indirectly as a targeting or delivery mechanism
for bringing a pharmaceutical agent to cells or tissue which
express IRS-p53h.
[0124] In another embodiment, a vector expressing the complement of
the polynucleotide encoding IRS-p53h may be administered to a
subject to treat or prevent cancer, including but not limited to
the cancers listed above.
[0125] In another embodiment, an antagonist of IRS-p53h may be
administered to a subject to prevent or treat an immunological
disorder. Such an immunological disorder may include, but is not
limited to, AIDS, Addison's disease, adult respiratory distress
syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,
asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune
thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus,
emphysema, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's thyroiditis, hypereosinophilia, irritable bowel
syndrome, lupus erythematosus, multiple sclerosis, myasthenia
gravis, myocardial or pericardial immunological disorders,
osteoarthritis, osteoporosis, pancreatitis, polymyositis,
rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
ulcerative colitis, Werner syndrome, and complications of cancer,
hemodialysis, and extracorporeal circulation; viral, bacterial,
fungal, parasitic, protozoal, and helminthic infections; and
trauma. In one aspect, antibodies which specifically bind IRS-p53h
may be used directly as an antagonist or indirectly as a targeting
or delivery mechanism for bringing a pharmaceutical agent to cells
or tissue which express IRS-p53h.
[0126] In another embodiment, a vector expressing the complement of
the polynucleotide encoding IRS-p53h may be administered to a
subject to treat or prevent an immune disorder including, but not
limited to, those listed above.
[0127] In other embodiments, any of the proteins, antagonists,
antibodies, agonists, complementary sequences, or vectors of the
invention may be administered in combination with other appropriate
therapeutic agents. Selection of the appropriate agents for use in
combination therapy may be made by one of ordinary skill in the
art, according to conventional pharmaceutical principles. The
combination of therapeutic agents may act synergistically to effect
the treatment or prevention of the various disorders described
above. Using this approach, one may be able to achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the
potential for adverse side effects.
[0128] An antagonist of IRS-p53h may be produced using methods
which are generally known in the art. In particular, purified
IRS-p53h may be used to produce antibodies or to screen libraries
of pharmaceutical agents to identify those which specifically bind
IRS-p53h. Antibodies to IRS-p53h may also be generated using
methods that are well known in the art. Such antibodies may
include, but are not limited to, polyclonal, monoclonal, chimeric,
and single chain antibodies, Fab fragments, and fragments produced
by a Fab expression library. Neutralizing antibodies (i.e., those
which inhibit dimer formation) are especially preferred for
therapeutic use.
[0129] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others may be immunized by
injection with IRS-p53h or with any fragment or oligopeptide
thereof which has immunogenic properties. Depending on the host
species, various adjuvants may be used to increase immunological
response. Such adjuvants include, but are not limited to, Freund's,
mineral gels such as aluminum hydroxide, and surface active
substances such as lysolecithin, pluronic polyols, polyanions,
peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants
used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium
parvum are especially preferable.
[0130] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to IRS-p53h have an amino acid
sequence consisting of at least about 5 amino acids, and, more
preferably, of at least about 10 amino acids. It is also preferable
that these oligopeptides, peptides, or fragments are identical to a
portion of the amino acid sequence of the natural protein and
contain the entire amino acid sequence of a small, naturally
occurring molecule. Short stretches of IRS-p53h amino acids may be
fused with those of another protein, such as KLH, and antibodies to
the chimeric molecule may be produced.
[0131] Monoclonal antibodies to IRS-p53h may be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G.
et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl.
Acad. Sci. 80:2026-2030; and Cole, S. P. et al. (1984) Mol. Cell
Biol. 62:109-120.)
[0132] In addition, techniques developed for the production of
"chimeric antibodies," such as the splicing of mouse antibody genes
to human antibody genes to obtain a molecule with appropriate
antigen specificity and biological activity, can be used. (See,
e.g., Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci.
81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; and
Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively,
techniques described for the production of single chain antibodies
may be adapted, using methods known in the art, to produce
IRS-p53h-specific single chain antibodies. Antibodies with related
specificity, but of distinct idiotypic composition, may be
generated by chain shuffling from random combinatorial
immunoglobulin libraries. (See, e.g., Burton D. R. (1991) Proc.
Natl. Acad. Sci. 88:10134-10137.)
[0133] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the literature. (See, e.g., Orlandi, R. et
al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; and Winter, G. et
al. (1991) Nature 349:293-299.)
[0134] Antibody fragments which contain specific binding sites for
IRS-p53h may also be generated. For example, such fragments
include, but are not limited to, F(ab')2 fragments produced by
pepsin digestion of the antibody molecule and Fab fragments
generated by reducing the disulfide bridges of the F(ab')2
fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity. (See, e.g., Huse, W. D.
et al. (1989) Science 246:1275-1281.)
[0135] Various immunoassays may be used for screening to identify
antibodies having the desired specificity. Numerous protocols for
competitive binding or immunoradiometric assays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art. Such immunoassays typically involve the
measurement of complex formation between IRS-p53h and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering IRS-p53h
epitopes is preferred, but a competitive binding assay may also be
employed. (Maddox, supra.)
[0136] In another embodiment of the invention, the polynucleotides
encoding IRS-p53h, or any fragment or complement thereof, may be
used for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding IRS-p53h may be used in situations in which
it would be desirable to block the transcription of the mRNA. In
particular, cells may be transformed with sequences complementary
to polynucleotides encoding IRS-p53h. Thus, complementary molecules
or fragments may be used to modulate IRS-p53h activity, or to
achieve regulation of gene function. Such technology is now well
known in the art, and sense or antisense oligonucleotides or larger
fragments can be designed from various locations along the coding
or control regions of sequences encoding IRS-p53h.
[0137] Expression vectors derived from retroviruses, adenoviruses,
or herpes or vaccinia viruses, or from various bacterial plasmids,
may be used for delivery of nucleotide sequences to the targeted
organ, tissue, or cell population. Methods which are well known to
those skilled in the art can be used to construct vectors which
will express nucleic acid sequences complementary to the
polynucleotides of the gene encoding IRS-p53h. (See, e.g.,
Sambrook, supra; and Ausubel, supra.)
[0138] Genes encoding IRS-p53h can be turned off by transforming a
cell or tissue with expression vectors which express high levels of
a polynucleotide, or fragment thereof, encoding IRS-p53h. Such
constructs may be used to introduce untranslatable sense or
antisense sequences into a cell. Even in the absence of integration
into the DNA, such vectors may continue to transcribe RNA molecules
until they are disabled by endogenous nucleases. Transient
expression may last for a month or more with a non-replicating
vector, and may last even longer if appropriate replication
elements are part of the vector system.
[0139] As mentioned above, modifications of gene expression can be
obtained by designing complementary sequences or antisense
molecules (DNA, RNA, or PNA) to the control, 5', or regulatory
regions of the gene encoding IRS-p53h. Oligonucleotides derived
from the transcription initiation site, e.g., between about
positions -10 and +10 from the start site, are preferred.
Similarly, inhibition can be achieved using triple helix
base-pairing methodology. Triple helix pairing is useful because it
causes inhibition of the ability of the double helix to open
sufficiently for the binding of polymerases, transcription factors,
or regulatory molecules. Recent therapeutic advances using triplex
DNA have been described in the literature. (See, e.g., Gee, J. E.
et al. (1994) in Huber, B. E. and B. I. Carr, Molecular and
Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., pp.
163-177.) A complementary sequence or antisense molecule may also
be designed to block translation of mRNA by preventing the
transcript from binding to ribosomes.
[0140] Ribozymes, enzymatic RNA molecules, may also be used to
catalyze the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridization of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic
cleavage. For example, engineered hammerhead motif ribozyme
molecules may specifically and efficiently catalyze endonucleolytic
cleavage of sequences encoding IRS-p53h.
[0141] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites, including the following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15
and 20 ribonucleotides, corresponding to the region of the target
gene containing the cleavage site, may be evaluated for secondary
structural features which may render the oligonucleotide
inoperable. The suitability of candidate targets may also be
evaluated by testing accessibility to hybridization with
complementary oligonucleotides using ribonuclease protection
assays.
[0142] Complementary ribonucleic acid molecules and ribozymes of
the invention may be prepared by any method known in the art for
the synthesis of nucleic acid molecules. These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis. Alternatively, RNA molecules
may be generated by in vitro and in vivo transcription of DNA
sequences encoding IRS-p53h. Such DNA sequences may be incorporated
into a wide variety of vectors with suitable RNA polymerase
promoters such as T7 or SP6. Alternatively, these cDNA constructs
that synthesize complementary RNA, constitutively or inducibly, can
be introduced into cell lines, cells, or tissues.
[0143] RNA molecules may be modified to increase intracellular
stability and half-life. Possible modifications include, but are
not limited to, the addition of flanking sequences at the 5' and/or
3' ends of the molecule, or the use of phosphorothioate or 2'
O-methyl rather than phosphodiesterase linkages within the backbone
of the molecule. This concept is inherent in the production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine, queosine, and wybutosine, as
well as acetyl-, methyl-, thio-, and similarly modified forms of
adenine, cytidine, guanine, thymine, and uridine which are not as
easily recognized by endogenous endonucleases.
[0144] Many methods for introducing vectors into cells or tissues
are available and equally suitable for use in vivo, in vitro, and
ex vivo. For ex vivo therapy, vectors may be introduced into stem
cells taken from the patient and clonally propagated for autologous
transplant back into that same patient. Delivery by transfection,
by liposome injections, or by polycationic amino polymers may be
achieved using methods which are well known in the art. (See, e.g.,
Goldman, C. K. et al. (1997) Nature Biotechnology 15:462-466.)
[0145] Any of the therapeutic methods described above may be
applied to any subject in need of such therapy, including, for
example, mammals such as dogs, cats, cows, horses, rabbits,
monkeys, and most preferably, humans.
[0146] An additional embodiment of the invention relates to the
administration of a pharmaceutical or sterile composition, in
conjunction with a pharmaceutically acceptable carrier, for any of
the therapeutic effects discussed above. Such pharmaceutical
compositions may consist of IRS-p53h, antibodies to IRS-p53h, and
mimetics, agonists, antagonists, or inhibitors of IRS-p53h. The
compositions may be administered alone or in combination with at
least one other agent, such as a stabilizing compound, which may be
administered in any sterile, biocompatible pharmaceutical carrier
including, but not limited to, saline, buffered saline, dextrose,
and water. The compositions may be administered to a patient alone,
or in combination with other agents, drugs, or hormones.
[0147] The pharmaceutical compositions utilized in this invention
may be administered by any number of routes including, but not
limited to, oral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, or rectal means.
[0148] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically-acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Further details on techniques for
formulation and administration may be found in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing Co.,
Easton, Pa.).
[0149] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the patient.
[0150] Pharmaceutical preparations for oral use can be obtained
through combining active compounds with solid excipient and
processing the resultant mixture of granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries
can be added, if desired. Suitable excipients include carbohydrate
or protein fillers, such as sugars, including lactose, sucrose,
mannitol, and sorbitol; starch from corn, wheat, rice, potato, or
other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
gums, including arabic and tragacanth; and proteins, such as
gelatin and collagen. If desired, disintegrating or solubilizing
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, and alginic acid or a salt thereof, such as
sodium alginate.
[0151] Dragee cores may be used in conjunction with suitable
coatings, such as concentrated sugar solutions, which may also
contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for product
identification or to characterize the quantity of active compound,
i.e., dosage.
[0152] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers
or binders, such as lactose or starches, lubricants, such as talc
or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
[0153] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks's solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils, such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the
suspension may also contain suitable stabilizers or agents to
increase the solubility of the compounds and allow for the
preparation of highly concentrated solutions.
[0154] For topical or nasal administration, penetrants appropriate
to the particular barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.
[0155] The pharmaceutical compositions of the present invention may
be manufactured in as manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilizing processes.
[0156] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and
succinic acid. Salts tend to be more soluble in aqueous or other
protonic solvents than are the corresponding free base forms. In
other cases, the preferred preparation may be a lyophilized powder
which may contain any or all of the following: 1 mM to 50 mM
histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, at a pH range
of 4.5 to 5.5, that is combined with buffer prior to use.
[0157] After pharmaceutical compositions have been prepared, they
can be placed in an appropriate container and labeled for treatment
of an indicated condition. For administration of IRS-p53h, such
labeling would include amount, frequency, and method of
administration.
[0158] Pharmaceutical compositions suitable for use in the
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
The determination of an effective dose is well within the
capability of those skilled in the art.
[0159] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays, e.g., of
neoplastic cells or in animal models such as mice, rats, rabbits,
dogs, or pigs. An animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans.
[0160] A therapeutically effective dose refers to that amount of
active ingredient, for example IRS-p53h or fragments thereof,
antibodies of IRS-p53h, and agonists, antagonists or inhibitors of
IRS-p53h, which ameliorates the symptoms or condition. Therapeutic
efficacy and toxicity may be determined by standard pharmaceutical
procedures in cell cultures or with experimental animals, such as
by calculating the ED.sub.50 (the dose therapeutically effective in
50% of the population) or LD.sub.50 (the dose lethal to 50% of the
population) statistics. The dose ratio of therapeutic to toxic
effects is the therapeutic index, and it can be expressed as the
ED.sub.50/LD50 ratio. Pharmaceutical compositions which exhibit
large therapeutic indices are preferred. The data obtained from
cell culture assays and animal studies are used to formulate a
range of dosage for human use. The dosage contained in such
compositions is preferably within a range of circulating
concentrations that includes the ED.sub.50 with little or no
toxicity. The dosage varies within this range depending upon the
dosage form employed, the sensitivity of the patient, and the route
of administration.
[0161] The exact dosage will be determined by the practitioner, in
light of factors related to the subject requiring treatment. Dosage
and administration are adjusted to provide sufficient levels of the
active moiety or to maintain the desired effect. Factors which may
be taken into account include the severity of the disease state,
the general health of the subject, the age, weight, and gender of
the subject, time and frequency of administration, drug
combination(s), reaction sensitivities, and response to therapy.
Long-acting pharmaceutical compositions may be administered every 3
to 4 days, every week, or biweekly depending on the half-life and
clearance rate of the particular formulation.
[0162] Normal dosage amounts may vary from about 0.1 .mu.g to
100,000 .mu.g, up to a total dose of about 1 gram, depending upon
the route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature and generally
available to practitioners in the art. Those skilled in the art
will employ different formulations for nucleotides than for
proteins or their inhibitors. Similarly, delivery of
polynucleotides or polypeptides will be specific to particular
cells, conditions, locations, etc.
DIAGNOSTICS
[0163] In another embodiment, antibodies which specifically bind
IRS-p53h may be used for the diagnosis of disorders characterized
by expression of IRS-p53h, or in assays to monitor patients being
treated with IRS-p53h or agonists, antagonists, or inhibitors of
IRS-p53h. Antibodies useful for diagnostic purposes may be prepared
in the same manner as described above for therapeutics. Diagnostic
assays for IRS-p53h include methods which utilize the antibody and
a label to detect IRS-p53h in human body fluids or in extracts of
cells or tissues. The antibodies may be used with or without
modification, and may be labeled by covalent or non-covalent
attachment of a reporter molecule. A wide variety of reporter
molecules, several of which are described above, are known in the
art and may be used.
[0164] A variety of protocols for measuring IRS-p53h, including
ELISAs, RIAs, and FACS, are known in the art and provide a basis
for diagnosing altered or abnormal levels of IRS-p53h expression.
Normal or standard values for IRS-p53h expression are established
by combining body fluids or cell extracts taken from normal
mammalian subjects, preferably human, with antibody to IRS-p53h
under conditions suitable for complex formation The amount of
standard complex formation may be quantitated by various methods,
preferably by photometric means. Quantities of IRS-p53h expressed
in subject, control, and disease samples from biopsied tissues are
compared with the standard values. Deviation between standard and
subject values establishes the parameters for diagnosing
disease.
[0165] In another embodiment of the invention, the polynucleotides
encoding IRS-p53h may be used for diagnostic purposes. The
polynucleotides which may be used include oligonucleotide
sequences, complementary RNA and DNA molecules, and PNAs. The
polynucleotides may be used to detect and quantitate gene
expression in biopsied tissues in which expression of IRS-p53h may
be correlated with disease. The diagnostic assay may be used to
determine absence, presence, and excess expression of IRS-p53h, and
to monitor regulation of IRS-p53h levels during therapeutic
intervention.
[0166] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding IRS-p53h or closely related molecules may be
used to identify nucleic acid sequences which encode IRS-p53h. The
specificity of the probe, whether it is made from a highly specific
region, e.g., the 5' regulatory region, or from a less specific
region, e.g., a conserved motif, and the stringency of the
hybridization or amplification (maximal, high, intermediate, or
low), will determine whether the probe identifies only naturally
occurring sequences encoding IRS-p53h, alleles, or related
sequences.
[0167] Probes may also be used for the detection of related
sequences, and should preferably have at least 50% sequence
identity to any of the IRS-p53h encoding sequences. The
hybridization probes of the subject invention may be DNA or RNA and
may be derived from the sequence of SEQ ID NO:2, and SEQ ID NO:4,
or from genomic sequences including promoters, enhancers, and
introns of the IRS-p53h gene.
[0168] Means for producing specific hybridization probes for DNAs
encoding IRS-p53h include the cloning of polynucleotide sequences
encoding IRS-p53h or IRS-p53h derivatives into vectors for the
production of mRNA probes. Such vectors are known in the art, are
commercially available, and may be used to synthesize RNA probes in
vitro by means of the addition of the appropriate RNA polymerases
and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a variety of reporter groups, for example, by
radionuclides such as .sup.32P or .sup.35S, or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and the like.
[0169] Polynucleotide sequences encoding IRS-p53h may be used for
the diagnosis of a disorder associated with expression of IRS-p53h.
Examples of such a disorder include, but are not limited to, a
disorder associated with insulin response such as type 2
(non-insulin-dependent) diabetes mellitus, hyperglycemia, myotonic
muscular dystrophy, acanthosis nigricans, retinopathy, nephropathy,
atherosclerotic coronary and peripheral arterial disease, and
peripheral and autonomic neuropathies; a reproductive disorder such
as, disorders of prolactin production; infertility, including tubal
disease, ovulatory defects, and endometriosis; disruptions of the
estrous cycle, disruptions of the menstrual cycle, polycystic ovary
syndrome, ovarian hyperstimulation syndrome, endometrial and
ovarian tumors, autoimmune disorders, ectopic pregnancy, and
teratogenesis; cancer of the breast, uterine fibroids, fibrocystic
breast disease, galactorrhea; disruptions of spermatogenesis,
abnormal sperm physiology, cancer of the testis, cancer of the
prostate, benign prostatic hyperplasia, prostatitis, Peyronie's
disease, carcinoma of the male breast and gynecomastia; Alzheimer's
disease; a cancer such as, adenocarcinoma, leukemia, lymphoma,
melanoma, myeloma, sarcoma, and teratocarcinoma, and particularly
cancers of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall bladder, ganglia, gastrointestinal tract,
heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid,
penis, prostate, salivary glands, skin, spleen, testis, thymus,
thyroid, and uterus; and an immunological disorder such as AIDS,
Addison's disease, adult respiratory distress syndrome, allergies,
ankylosing spondylitis, amyloidosis, anemia, asthma,
atherosclerosis, autoimmune hemolytic anemia, autoimmune
thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus,
emphysema, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's thyroiditis, hypereosinophilia, irritable bowel
syndrome, lupus erythematosus, multiple sclerosis, myasthenia
gravis, myocardial or pericardial immunological disorders,
osteoarthritis, osteoporosis, pancreatitis, polymyositis,
rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
ulcerative colitis, Werner syndrome, and complications of cancer,
hemodialysis, and extracorporeal circulation; viral, bacterial,
fungal, parasitic, protozoal, and helminthic infections; and
trauma. The polynucleotide sequences encoding IRS-p53h may be used
in Southern or northern analysis, dot blot, or other membrane-based
technologies; in PCR technologies; in dipstick, pin, and ELISA
assays; and in microarrays utilizing fluids or tissues from
patients to detect altered IRS-p53h expression. Such qualitative or
quantitative methods are well known in the art.
[0170] In a particular aspect, the nucleotide sequences encoding
IRS-p53h may be useful in assays that detect the presence of
associated disorders, particularly those mentioned above. The
nucleotide sequences encoding IRS-p53h may be labeled by standard
methods and added to a fluid or tissue sample from a patient under
conditions suitable for the formation of hybridization complexes.
After a suitable incubation period, the sample is washed and the
signal is quantitated and compared with a standard value. If the
amount of signal in the patient sample is significantly altered in
comparison to a control sample then the presence of altered levels
of nucleotide sequences encoding IRS-p53h in the sample indicates
the presence of the associated disorder. Such assays may also be
used to evaluate the efficacy of a particular therapeutic treatment
regimen in animal studies, in clinical trials, or to monitor the
treatment of an individual patient.
[0171] In order to provide a basis for the diagnosis of a disorder
associated with expression of IRS-p53h, a normal or standard
profile for expression is established. This may be accomplished by
combining body fluids or cell extracts taken from normal subjects,
either animal or human, with a sequence, or a fragment thereof,
encoding IRS-p53h, under conditions suitable for hybridization or
amplification. Standard hybridization may be quantified by
comparing the values obtained from normal subjects with values from
an experiment in which a known amount of a substantially purified
polynucleotide is used. Standard values obtained in this manner may
be compared with values obtained from samples from patients who are
symptomatic for a disorder. Deviation from standard values is used
to establish the presence of a disorder.
[0172] Once the presence of a disorder is established and a
treatment protocol is initiated, hybridization assays may be
repeated on a regular basis to determine if the level of expression
in the patient begins to approximate that which is observed in the
normal subject. The results obtained from successive assays may be
used to show the efficacy of treatment over a period ranging from
several days to months.
[0173] With respect to cancer, the presence of a relatively high
amount of transcript in biopsied tissue from an individual may
indicate a predisposition for the development of the disease, or
may provide a means for detecting the disease prior to the
appearance of actual clinical symptoms. A more definitive diagnosis
of this type may allow health professionals to employ preventative
measures or aggressive treatment earlier thereby preventing the
development or further progression of the cancer.
[0174] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding IRS-p53h may involve the use of PCR.
These oligomers may be chemically synthesized, generated
enzymatically, or produced in vitro. Oligomers will preferably
contain a fragment of a polynucleotide encoding IRS-p53h, or a
fragment of a polynucleotide complementary to the polynucleotide
encoding IRS-p53h, and will be employed under optimized conditions
for identification of a specific gene or condition. Oligomers may
also be employed under less stringent conditions for detection or
quantitation of closely related DNA or RNA sequences.
[0175] Methods which may also be used to quantitate the expression
of IRS-p53h include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and interpolating
results from standard curves. (See, e.g., Melby, P. C. et al.
(1993) J. Immunol. Methods 159:235-244; and Duplaa, C. et al.
(1993) Anal. Biochem. 229-236.) The speed of quantitation of
multiple samples may be accelerated by running the assay in an
ELISA format where the oligomer of interest is presented in various
dilutions and a spectrophotometric or colorimetric response gives
rapid quantitation.
[0176] In further embodiments, oligonucleotides or longer fragments
derived from any of the polynucleotide sequences described herein
may be used as targets in a microarray. The microarray can be used
to monitor the expression level of large numbers of genes
simultaneously and to identify genetic variants, mutations, and
polymorphisms. This information may be used to determine gene
function, to understand the genetic basis of a disorder, to
diagnose a disorder, and to develop and monitor the activities of
therapeutic agents.
[0177] Microarrays may be prepared, used, and analyzed using
methods known in the art. (See, e.g., Brennan, T. M. et al. (1995)
U.S. Pat. No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad.
Sci. 93:10614-10619; Baldeschweiler et al. (1995) PCT application
WO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;
Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155;
and Heller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)
[0178] In another embodiment of the invention, nucleic acid
sequences encoding IRS-p53h may be used to generate hybridization
probes useful in mapping the naturally occurring genomic sequence.
The sequences may be mapped to a particular chromosome, to a
specific region of a chromosome, or to artificial chromosome
constructions, e.g., human artificial chromosomes (HACs), yeast
artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial P1 constructions, or single chromosome cDNA
libraries. (See, e.g., Price, C. M. (1993) Blood Rev. 7:127-134;
and Trask, B. J. (1991) Trends Genet. 7:149-154.)
[0179] Fluorescent in situ hybridization (FISH) may be correlated
with other physical chromosome mapping techniques and genetic map
data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, R. A.
(ed.) Molecular Biology and Biotechnology, VCH Publishers New York,
N.Y., pp. 965-968.) Examples of genetic map data can be found in
various scientific journals or at the Online Mendelian Inheritance
in Man (OMIM) site. Correlation between the location of the gene
encoding IRS-p53h on a physical chromosomal map and a specific
disorder, or a predisposition to a specific disorder, may help
define the region of DNA associated with that disorder. The
nucleotide sequences of the invention may be used to detect
differences in gene sequences among normal, carrier, and affected
individuals.
[0180] In situ hybridization of chromosomal preparations and
physical mapping techniques, such as linkage analysis using
established chromosomal markers, may be used for extending genetic
maps. Often the placement of a gene on the chromosome of another
mammalian species, such as mouse, may reveal associated markers
even if the number or arm of a particular human chromosome is not
known. New sequences can be assigned to chromosomal arms by
physical mapping. This provides valuable information to
investigators searching for disease genes using positional cloning
or other gene discovery techniques. Once the disease or syndrome
has been crudely localized by genetic linkage to a particular
genomic region, e.g., AT to 11q22-23, any sequences mapping to that
area may represent associated or regulatory genes for further
investigation. (See, e.g., Gatti, R. A. et al. (1988) Nature
336:577-580.) The nucleotide sequence of the subject invention may
also be used to detect differences in the chromosomal location due
to translocation, inversion, etc., among normal, carrier, or
affected individuals.
[0181] In another embodiment of the invention, IRS-p53h, its
catalytic or immunogenic fragments, or oligopeptides thereof can be
used for screening libraries of compounds in any of a variety of
drug screening techniques. The fragment employed in such screening
may be free in solution, affixed to a solid support, borne on a
cell surface, or located intracellularly. The formation of binding
complexes between IRS-p53h and the agent being tested may be
measured.
[0182] Another technique for drug screening provides for high
throughput screening of compounds having suitable binding affinity
to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT
application WO84/03564.) In this method, large numbers of different
small test compounds are synthesized on a solid substrate, such as
plastic pins or some other surface. The test compounds are reacted
with IRS-p53h, or fragments thereof, and washed. Bound IRS-p53h is
then detected by methods well known in the art. Purified IRS-p53h
can also be coated directly onto plates for use in the
aforementioned drug screening techniques. Alternatively,
non-neutralizing antibodies can be used to capture the peptide and
immobilize it on a solid support.
[0183] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding IRS-p53h specifically compete with a test compound for
binding IRS-p53h. In this manner, antibodies can be used to detect
the presence of any peptide which shares one or more antigenic
determinants with IRS-p53h.
[0184] In additional embodiments, the nucleotide sequences which
encode IRS-p53h may be used in any molecular biology techniques
that have yet to be developed, provided the new techniques rely on
properties of nucleotide sequences that are currently known,
including, but not limited to, such properties as the triplet
genetic code and specific base pair interactions.
[0185] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0186] I cDNA Library Construction
BRSTNOT04
[0187] The BRSTNOT04 cDNA library was constructed from
microscopically normal breast tissue removed from a 62-year-old
female (specimen #0116A) during unilateral extended simple
mastectomy following diagnosis of invasive grade 3 (of 4), nuclear
grade 2 (of 3) mammary ductal carcinoma. The surgical margins were
found negative for tumor. Also, a 0.4 cm focus of in situ carcinoma
was identified in the lower quadrant of the breast. Prior to
surgery, the patient was diagnosed with benign hypertension,
cerebrovascular disease, atherosclerosis, hyperlipidemia, and
hematuria. The patient family history included liver cancer in a
sibling.
[0188] The frozen tissue was homogenized and lysed using a
Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Instruments,
Westbury, N.J.) in guanidinium isothiocyanate solution. The lysate
was centrifuged over a 5.7 M CsCl cushion using an Beckman SW28
rotor in a Beckman L8-70M Ultracentrifuge (Beckman Instruments) for
18 hours at 25,000 rpm at ambient temperature. The RNA was
extracted with acid phenol pH 4.0, precipitated using 0.3 M sodium
acetate and 2.5 volumes of ethanol, resuspended in RNAse-free
water, and treated with DNase at 37.degree. C. RNA extraction and
precipitation were repeated as before. The mRNA was then isolated
with the Qiagen Oligotex kit (QIAGEN, Inc.; Chatsworth, Calif.) and
used to construct the cDNA library.
[0189] The mRNA was handled according to the recommended protocols
in the SuperScript plasmid system (Catalog #18248-013; GIBCO-BRL).
cDNA synthesis was initiated with a NotI-oligo d(T) primer.
Double-stranded cDNA was blunted, ligated to SalI adaptors,
digested with NotI, fractionated on a Sepharose CL4B column
(Catalog #275105-01; Pharmacia Upjohn), and those cDNAs exceeding
400 bp were ligated into the NotI and SalI sites of the vector
pSport I. The plasmid pSport I was subsequently transformed into
DH5.alpha..TM. competent cells (Catalog #18258-012; GIBCO-BRL).
ADRETUT05
[0190] The ADRETUT05 cDNA library was constructed from tumor tissue
obtained from the right adrenal gland of a 52 year-old Caucasian
female (specimen #0058) during a unilateral adrenalectomy.
Pathology indicated a pheochromocytoma. Patient history included
benign hypertension, depressive disorder, chronic sinusitis,
idiopathic proctocolitis, urinary tract infection and irritable
colon. Family history included benign hypertension in a sibling,
cerebrovascular disease, secondary Parkinsonism, and irritable
colon in the mother; atherosclerotic coronary artery disease,
hyperlipidemia and malignant brain neoplasm in siblings, and
secondary Parkinsonism in the father.
[0191] The frozen tissue was homogenized and lysed in Trizol
reagent (1 g tissue/10 ml Trizol; Catalog # 10296-028; GIBco-BRL),
a monoplastic solution of phenol and guanidine isothiocyanate,
using a Brinkmann Homogenizer Polytron PT-3000 (Brinkmann
Instruments, Westbury, N.Y.). After a brief incubation on ice,
chloroform was added (1:5 v/v) and the lysate was centrifuged. The
upper layer was removed to a fresh tube and the RNA extracted with
isopropanol, resuspended in DEPC-treated water, and treated with
DNase for 25 minutes at 37.degree. C. RNA was extracted and
precipitated as before. The mRNA was isolated using the Qiagen
Oligotex kit (QIAGEN, Inc., Chatsworth, Calif.) and used to
construct the cDNA library.
[0192] The mRNA was handled according to the recommended protocols
in the SuperScript plasmid system (Catalog # 18248-013, GIBCO-BRL).
cDNA synthesis was initiated with NotI-oligo d(T) primer.
Double-stranded cDNA was blunted, ligated to EcoRI adaptors,
digested with NotI, fractionated on a Sepharose CL4B column
(Catalog # 275105-01; Pharmacia Upjohn), and those cDNAs exceeding
400 bp were ligated into the NotI and EcoRI sites of the pINCY 1
vector (Incyte). The plasmid pINCY 1 was subsequently transformed
into DH5.alpha..TM. competent cells (Catalog # 18258-012;
GIBCO-BRL).
[0193] II Isolation and Sequencing of cDNA Clones
[0194] Plasmid DNA was released from the cells and purified using
the REAL Prep 96 plasmid kit (Catalog # 26173; QIAGEN, Inc.). The
recommended protocol was employed except for the following changes:
1) the bacteria were cultured in I ml of sterile Terrific Broth
(Catalog # 22711, GIBCO-BRL) with 25 mg/l carbenicillin, 0.4%
glycerol; 2) after inoculation, the cultures were incubated for 19
hours and at the end of incubation, the cells were lysed with 0.3
ml of lysis buffer; and 3) following isopropanol precipitation, the
plasmid DNA pellet was resuspended in 0.1 ml of distilled water.
After the last step in the protocol, samples were transferred to a
96-well block for storage at 4.degree. C.
[0195] The cDNAs were sequenced by the method of Sanger et al.
(1975; J. Mol. Biol. 94:441f), using a Hamilton Micro Lab 2200
(Hamilton, Reno, Nev.) in combination with Peltier Thermal Cyclers
(PTC200 from MJ Research, Watertown, Mass.) and Applied Biosystems
377 DNA Sequencing Systems.
[0196] III Homology Searching of cDNA Clones and Their Deduced
Proteins
[0197] The nucleotide sequences and/or amino acid sequences of the
Sequence Listing were used to query sequences in the GenBank,
SwissProt, BLOCKS, and Pima II databases. These databases, which
contain previously identified and annotated sequences, were
searched for regions of homology using BLAST (Basic Local Alignment
Search Tool). (See, e.g., Altschul, S. F. (1993) J. Mol. Evol
36:290-300; and Altschul et al. (1990) J. Mol. Biol.
215:403-410.)
[0198] BLAST produced alignments of both nucleotide and amino acid
sequences to determine sequence similarity. Because of the local
nature of the alignments, BLAST was especially useful in
determining exact matches or in identifying homologs which may be
of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant)
origin. Other algorithms could have been used when dealing with
primary sequence patterns and secondary structure gap penalties.
(See, e.g., Smith, T. et al. (1992) Protein Engineering 5:35-51.)
The sequences disclosed in this application have lengths of at
least 49 nucleotides and have no more than 12% uncalled bases
(where N is recorded rather than A, C, G, or T).
[0199] The BLAST approach searched for matches between a query
sequence and a database sequence. BLAST evaluated the statistical
significance of any matches found, and reported only those matches
that satisfy the user-selected threshold of significance. In this
application, threshold was set at 10.sup.-25 for nucleotides and
10.sup.-8 for peptides.
[0200] Incyte nucleotide sequences were searched against the
GenBank databases for primate (pri), rodent (rod), and other
mammalian sequences (mam), and deduced amino acid sequences from
the same clones were then searched against GenBank functional
protein databases, mammalian (mamp), vertebrate (vrtp), and
eukaryote (eukp), for homology.
[0201] Additionally, sequences identified from cDNA libraries may
be analyzed to identify those gene sequences encoding conserved
protein motifs using an appropriate analysis program, e.g., the
Block 2 Bioanalysis Program (Incyte, Palo Alto, Calif.). This motif
analysis program, based on sequence information contained in the
Swiss-Prot Database and PROSITE, is a method of determining the
function of uncharacterized proteins translated from genomic or
cDNA sequences. (See, e.g., Bairoch, A. et al. (1997) Nucleic Acids
Res. 25:217-221; and Attwood, T. K. et al. (1997) J. Chem. Inf.
Comput. Sci. 37:417-424.) PROSITE may be used to identify common
functional or structural domains in divergent proteins. The method
is based on weight matrices. Motifs identified by this method are
then calibrated against the SWISS-PROT database in order to obtain
a measure of the chance distribution of the matches.
[0202] In another alternative, Hidden Markov models (HMMs) may be
used to find protein domains, each defined by a dataset of proteins
known to have a common biological function. (See, e.g., Pearson, W.
R. and D. J. Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; and
Smith, T. F. and M. S. Waterman (1981) J. Mol. Biol. 147:195-197.)
HMMs were initially developed to examine speech recognition
patterns, but are now being used in a biological context to analyze
protein and nucleic acid sequences as well as to model protein
structure. (See, e.g., Krogh, A. et al. (1994) J. Mol. Biol.
235:1501-1531; and Collin, M. et al. (1993) Protein Sci.
2:305-314.) HMMs have a formal probabilistic basis and use
position-specific scores for amino acids or nucleotides. The
algorithm continues to incorporate information from newly
identified sequences to increase its motif analysis
capabilities.
[0203] IV. Northern Analysis
[0204] Northern analysis is a laboratory technique used to detect
the presence of a transcript of a gene and involves the
hybridization of a labeled nucleotide sequence to a membrane on
which RNAs from a particular cell type or tissue have been bound.
(See, e.g., Sambrook, supra, ch. 7; and Ausubel, supra, ch. 4 and
16.)
[0205] Analogous computer techniques applying BLAST are used to
search for identical or related molecules in nucleotide databases
such as GenBank or LIFESEQ.TM. database (Incyte Pharmaceuticals).
This analysis is much faster than multiple membrane-based
hybridizations. In addition, the sensitivity of the computer search
can be modified to determine whether any particular match is
categorized as exact or homologous.
[0206] The basis of the search is the product score, which is
defined as:
% sequence identity.times.% maximum BLAST score/100
[0207] The product score takes into account both the degree of
similarity between two sequences and the length of the sequence
match. For example, with a product score of 40, the match will be
exact within a 1% to 2% error, and, with a product score of 70, the
match will be exact. Homologous molecules are usually identified by
selecting those which show product scores between 15 and 40,
although lower scores may identify related molecules.
[0208] The results of northern analysis are reported as a list of
libraries in which the transcript encoding IRS-p53h occurs.
Abundance and percent abundance are also reported. Abundance
directly reflects the number of times a particular transcript is
represented in a cDNA library, and percent abundance is abundance
divided by the total number of sequences examined in the cDNA
library.
[0209] V. Extension of IRS-p53h Encoding Polynucleotides
[0210] The nucleic acid sequences of Incyte Clones 918158 and
2493150 were used to design oligonucleotide primers for extending
partial nucleotide sequences to full length. For each nucleic acid
sequence, one primer was synthesized to initiate extension of an
antisense polynucleotide, and the other was synthesized to initiate
extension of a sense polynucleotide. Primers were used to
facilitate the extension of the known sequence "outward" generating
amplicons containing new unknown nucleotide sequence for the region
of interest. The initial primers were designed from the cDNA using
OLIGO 4.06 (National Biosciences, Plymouth, Minn.), or another
appropriate program, to be about 22 to 30 nucleotides in length, to
have a GC content of about 50% or more, and to anneal to the target
sequence at temperatures of about 68.degree. C. to about 72.degree.
C. Any stretch of nucleotides which would result in hairpin
structures and primer-primer dimerizations was avoided.
[0211] Selected human cDNA libraries (GIBCO/BRL) were used to
extend the sequence. If more than one extension is necessary or
desired, additional sets of primers are designed to further extend
the known region.
[0212] High fidelity amplification was obtained by following the
instructions for the XL-PCR kit (Perkin Elmer) and thoroughly
mixing the enzyme and reaction mix. PCR was performed using the
Peltier Thermal Cycler (PTC200; M. J. Research, Watertown, Mass.),
beginning with 40 pmol of each primer and the recommended
concentrations of all other components of the kit, with the
following parameters:
1 Step 1 94.degree. C. for 1 min (initial denaturation) Step 2
65.degree. C. for 1 min Step 3 68.degree. C. for 6 min Step 4
94.degree. C. for 15 sec Step 5 65.degree. C. for 1 min Step 6
68.degree. C. for 7 min Step 7 Repeat steps 4 through 6 for an
additional 15 cycles Step 8 94.degree. C. for 15 sec Step 9
65.degree. C. for 1 min Step 10 68.degree. C. for 7:15 min Step 11
Repeat steps 8 through 10 for an additional 12 cycles Step 12
72.degree. C. for 8 min Step 13 4.degree. C. (and holding)
[0213] A 5 .mu.l to 10 .mu.l aliquot of the reaction mixture was
analyzed by electrophoresis on a low concentration (about 0.6% to
0.8%) agarose mini-gel to determine which reactions were successful
in extending the sequence. Bands thought to contain the largest
products were excised from the gel, purified using QIAQuick.TM.
(QIAGEN Inc.), and trimmed of overhangs using Klenow enzyme to
facilitate religation and cloning.
[0214] After ethanol precipitation, the products were redissolved
in 13 .mu.l of ligation buffer, 1 .mu.l T4-DNA ligase (15 units)
and 1 .mu.l T4 polynucleotide kinase were added, and the mixture
was incubated at room temperature for 2 to 3 hours, or overnight at
16.degree. C. Competent E. coli cells (in 40 .mu.l of appropriate
media) were transformed with 3 .mu.l of ligation mixture and
cultured in 80 .mu.l of SOC medium. (See, e.g., Sambrook, supra,
Appendix A, p. 2.) After incubation for one hour at 37.degree. C.,
the E. coli mixture was plated on Luria Bertani (LB) agar (See,
e.g., Sambrook, supra, Appendix A, p. 1) containing carbenicillin
(2.times. carb). The following day, several colonies were randomly
picked from each plate and cultured in 150 .mu.l of liquid
LB/2.times. Carb medium placed in an individual well of an
appropriate commercially-available sterile 96-well microtiter
plate. The following day, 5 .mu.l of each overnight culture was
transferred into a non-sterile 96-well plate and, after dilution
1:10 with water, 5 .mu.l from each sample was transferred into a
PCR array.
[0215] For PCR amplification, 18 .mu.l of concentrated PCR reaction
mix (3.3.times.) containing 4 units of rTth DNA polymerase, a
vector primer, and one or both of the gene specific primers used
for the extension reaction were added to each well. Amplification
was performed using the following conditions:
2 Step 1 94.degree. C. for 60 sec Step 2 94.degree. C. for 20 sec
Step 3 55.degree. C. for 30 sec Step 4 72.degree. C. for 90 sec
Step 5 Repeat steps 2 through 4 for an additional 29 cycles Step 6
72.degree. C. for 180 sec Step 7 4.degree. C. (and holding)
[0216] Aliquots of the PCR reactions were run on agarose gels
together with molecular weight markers. The sizes of the PCR
products were compared to the original partial cDNAs, and
appropriate clones were selected, ligated into plasmid, and
sequenced.
[0217] In like manner, the nucleotide sequences of SEQ ID NO:2, and
SEQ ID NO:4, are used to obtain 5' regulatory sequences using the
procedure above, oligonucleotides designed for 5' extension, and an
appropriate genomic library.
[0218] VI. Labeling and Use of Individual Hybridization Probes
[0219] Hybridization probes derived from SEQ ID NO:2, and SEQ ID
NO:4, are employed to screen cDNAs, genomic DNAs, or mRNAs.
Although the labeling of oligonucleotides, consisting of about 20
base pairs, is specifically described, essentially the same
procedure is used with larger nucleotide fragments.
Oligonucleotides are designed using state-of-the-art software such
as OLIGO 4.06 (National Biosciences) and labeled by combining 50
pmol of each oligomer, 250 .mu.Ci of [.gamma.-.sup.32P] adenosine
triphosphate (Amersham, Chicago, Ill.), and T4 polynucleotide
kinase (DuPont NEN.RTM., Boston, Mass.). The labeled
oligonucleotides are substantially purified using a Sephadex G-25
superfine resin column (Pharmacia & Upjohn, Kalamazoo, Mich.).
An aliquot containing 10.sup.7 counts per minute of the labeled
probe is used in a typical membrane-based hybridization analysis of
human genomic DNA digested with one of the following endonucleases:
Ase I, Bgl II, Eco RI, Pst I, XbaI, or Pvu II (DuPont NEN, Boston,
Mass.).
[0220] The DNA from each digest is fractionated on a 0.7 percent
agarose gel and transferred to nylon membranes (Nytran Plus,
Schleicher & Schuell, Durham, N.H.). Hybridization is carried
out for 16 hours at 40.degree. C. To remove nonspecific signals,
blots are sequentially washed at room temperature under
increasingly stringent conditions up to 0.1.times. saline sodium
citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR.TM. film
(Kodak, Rochester, N.Y.) is exposed to the blots to film for
several hours, hybridization patterns are compared visually.
[0221] VII. Microarrays
[0222] A chemical coupling procedure and an ink jet device can be
used to synthesize array elements on the surface of a substrate.
(See, e.g., Baldeschweiler, supra.) An array analogous to a dot or
slot blot may also be used to arrange and link elements to the
surface of a substrate using thermal, UV, chemical, or mechanical
bonding procedures. A typical array may be produced by hand or
using available methods and machines and contain any appropriate
number of elements. After hybridization, nonhybridized probes are
removed and a scanner used to determine the levels and patterns of
fluorescence. The degree of complementarity and the relative
abundance of each probe which hybridizes to an element on the
microarray may be assessed through analysis of the scanned
images.
[0223] Full-length cDNAs, Expressed Sequence Tags (ESTs), or
fragments thereof may comprise the elements of the microarray.
Fragments suitable for hybridization can be selected using software
well known in the art such as LASERGENE.TM.. Full-length cDNAs,
ESTs, or fragments thereof corresponding to one of the nucleotide
sequences of the present invention, or selected at random from a
cDNA library relevant to the present invention, are arranged on an
appropriate substrate, e.g., a glass slide. The cDNA is fixed to
the slide using, e.g., UV cross-linking followed by thermal and
chemical treatments and subsequent drying. (See, e.g., Schena, M.
et al. (1995) Science 270:467-470; and Shalon, D. et al. (1996)
Genome Res. 6:639-645.) Fluorescent probes are prepared and used
for hybridization to the elements on the substrate. The substrate
is analyzed by procedures described above.
[0224] VIII. Complementary Polynucleotides
[0225] Sequences complementary to the IRS-p53h-encoding sequences,
or any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring IRS-p53h. Although use of
oligonucleotides comprising from about 15 to 30 base pairs is
described, essentially the same procedure is used with smaller or
with larger sequence fragments. Appropriate oligonucleotides are
designed using Oligo 4.06 software and the coding sequence of
IRS-p53h. To inhibit transcription, a complementary oligonucleotide
is designed from the most unique 5' sequence and used to prevent
promoter binding to the coding sequence. To inhibit translation, a
complementary oligonucleotide is designed to prevent ribosomal
binding to the IRS-p53h-encoding transcript.
[0226] IX. Expression of IRS-p53h
[0227] Expression of IRS-p53h is accomplished by subcloning the
cDNA into an appropriate vector and transforming the vector into
host cells. This vector contains an appropriate promoter, e.g.,
.beta.-galactosidase, upstream of the cloning site, operably
associated with the cDNA of interest. (See, e.g., Sambrook, supra,
pp. 404-433; and Rosenberg, M. et al. (1983) Methods Enzymol.
101:123-138.)
[0228] Induction of an isolated, transformed bacterial strain with
isopropyl beta-D-thiogalactopyranoside (IPTG) using standard
methods produces a fusion protein which consists of the first 8
residues of .beta.-galactosidase, about 5 to 15 residues of linker,
and the full length protein. The signal residues direct the
secretion of IRS-p53h into bacterial growth media which can be used
directly in the following assay for activity.
[0229] X Demonstration of IRS-p53h Activity
[0230] Human IR is expressed in and partially purified from chinese
hamster ovary cells or rat hepatoma cells as described by Yeh, et
al. (supra). IRS-p53h is incubated with the partially purified
human IR in the presence of 1 .mu.M insulin, 5 mM MgCl.sub.2, 5 mM
MnCl.sub.2, 0.5 mM ATP in 20 mM HEPES pH 7.5, 0.1% Triton X-100 for
20 min at 25.degree. C. The incubations are subjected to SDS-PAGE
electrophoresis (Sambrook, supra). Tyrosine-phosphorylated IRS-p53h
is detected by western blotting (Sambrook, supra) using a
horseradish peroxidase-conjugated anti-phosphotyrosine antibody
such as RC20 (Transduction Laboratories; Lexington, Ky.) and the
ECL detection system (Amersham International).
[0231] XI Production of IRS-p53h Specific Antibodies
[0232] IRS-p53h substantially purified using PAGE electrophoresis
(see, e.g., Harrington, M. G. (1990) Methods Enzymol. 182:488-495),
or other purification techniques, is used to immunize rabbits and
to produce antibodies using standard protocols.
[0233] Alternatively, the IRS-p53h amino acid sequence is analyzed
using LASERGENE.TM. software (DNASTAR Inc.) to determine regions of
high immunogenicity, and a corresponding oligopeptide is
synthesized and used to raise antibodies by means known to those of
skill in the art. Methods for selection of appropriate epitopes,
such as those near the C-terminus or in hydrophilic regions are
well described in the art. (See, e.g., Ausubel supra, ch. 11.)
[0234] Typically, oligopeptides 15 residues in length are
synthesized using an Applied Biosystems Peptide Synthesizer Model
431A using fmoc-chemistry and coupled to KLH (Sigma, St. Louis,
Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester
(MBS) to increase immunogenicity. (See, e.g., Ausubel supra.)
Rabbits are immunized with the oligopeptide-KLH complex in complete
Freund's adjuvant. Resulting antisera are tested for antipeptide
activity, for example, by binding the peptide to plastic, blocking
with 1% BSA, reacting with rabbit antisera, washing, and reacting
with radio-iodinated goat anti-rabbit IgG.
[0235] XII. Purification of Naturally Occurring IRS-p53h Using
Specific Antibodies
[0236] Naturally occurring or recombinant IRS-p53h is substantially
purified by immunoaffinity chromatography using antibodies specific
for IRS-p53h. An immunoaffinity column is constructed by covalently
coupling anti-IRS-p53h antibody to an activated chromatographic
resin, such as CNBr-activated Sepharose (Pharmacia & Upjohn).
After the coupling, the resin is blocked and washed according to
the manufacturer's instructions.
[0237] Media containing IRS-p53h are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of IRS-p53h (e.g., high ionic strength
buffers in the presence of detergent). The column is eluted under
conditions that disrupt antibody/IRS-p53h binding (e.g., a buffer
of pH 2 to pH 3, or a high concentration of a chaotrope, such as
urea or thiocyanate ion), and IRS-p53h is collected.
[0238] XIII. Identification of Molecules Which Interact with
IRS-p53h
[0239] IRS-p53h, or biologically active fragments thereof, are
labeled with .sup.125I Bolton-Hunter reagent. (See, e.g., Bolton et
al. (1973) Biochem. J. 133:529.) Candidate molecules previously
arrayed in the wells of a multi-well plate are incubated with the
labeled IRS-p53h, washed, and any wells with labeled IRS-p53h
complex are assayed. Data obtained using different concentrations
of IRS-p53h are used to calculate values for the number, affinity,
and association of IRS-p53h with the candidate molecules.
[0240] Various modifications and variations of the described
methods and systems of the invention will be apparent to those
skilled in the art without departing from the scope and spirit of
the invention. Although the invention has been described in
connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes for carrying out the invention which are
obvious to those skilled in molecular biology or related fields are
intended to be within the scope of the following claims.
Sequence CWU 1
1
* * * * *