U.S. patent application number 09/828423 was filed with the patent office on 2002-07-25 for growth-associated protease inhibitor heavy chain precursor.
This patent application is currently assigned to Incyte Pharmaceuticals, Inc.. Invention is credited to Guegler, Karl J., Hillman, Jennifer L., Patterson, Chandra.
Application Number | 20020099178 09/828423 |
Document ID | / |
Family ID | 22120329 |
Filed Date | 2002-07-25 |
United States Patent
Application |
20020099178 |
Kind Code |
A1 |
Hillman, Jennifer L. ; et
al. |
July 25, 2002 |
Growth-associated protease inhibitor heavy chain precursor
Abstract
The invention provides a human growth-associated protease
inhibitor heavy chain precursor (GAPIP) and polynucleotides which
identify and encode GAPIP. The invention also provides expression
vectors, host cells, antibodies, agonists, and antagonists. The
invention also provides methods for diagnosing, treating or
preventing disorders associated with expression of GAPIP.
Inventors: |
Hillman, Jennifer L.;
(Mountain View, CA) ; Guegler, Karl J.; (Menlo
Park, CA) ; Patterson, Chandra; (Mountain View,
CA) |
Correspondence
Address: |
INCYTE GENOMICS, INC.
PATENT DEPARTMENT
3160 Porter Drive
Palo Alto
CA
94304
US
|
Assignee: |
Incyte Pharmaceuticals,
Inc.
|
Family ID: |
22120329 |
Appl. No.: |
09/828423 |
Filed: |
April 5, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09828423 |
Apr 5, 2001 |
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09388774 |
Sep 2, 1999 |
|
|
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09388774 |
Sep 2, 1999 |
|
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09074579 |
May 7, 1998 |
|
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Current U.S.
Class: |
530/387.1 ;
424/178.1; 435/183; 435/193; 435/6.16; 435/69.1 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/811 20130101; A61P 35/00 20180101; A61P 43/00 20180101;
A61P 15/00 20180101; A61P 37/02 20180101 |
Class at
Publication: |
530/387.1 ;
435/183; 435/193; 435/6; 424/178.1; 435/69.1 |
International
Class: |
C07K 001/00 |
Claims
What is claimed is:
1. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of: a) an amino acid sequence of
SEQ ID NO:1, b) a naturally-occurring amino acid sequence having at
least 90% sequence identity to the sequence of SEQ ID NO:1, c) a
biologically-active fragment of the amino acid sequence of SEQ ID
NO:1, and d) an immunogenic fragment of the amino acid sequence of
SEQ ID NO:1.
2. An isolated polypeptide of claim 1, having a sequence of SEQ ID
NO:1.
3. An isolated antibody which specifically binds to a polypeptide
of claim 1.
4. A diagnostic test for a condition or disease associated with the
expression of GAPIP in a biological sample comprising the steps of:
a) combining the biological sample with an antibody of claim 3,
under conditions suitable for the antibody to bind the polypeptide
and form an antibody: polypeptide complex; and b) detecting the
complex, wherein the presence of the complex correlates with the
presence of the polypeptide in the biological sample.
5. The antibody of claim 3, wherein the antibody is: (a) a chimeric
antibody; (b) a single chain antibody; (c) a Fab fragment; (d) a
F(ab')2 fragment; or (e) a humanized antibody.
6. A composition comprising an antibody of claim 3 and an
acceptable excipient.
7. A method of diagnosing a condition or disease associated with
the expression of GAPIP in a subject, comprising administering to
said subject an effective amount of the composition of claim 6.
8. A composition of claim 6, wherein the antibody is labeled.
9. A method of diagnosing a condition or disease associated with
the expression of GAPIP in a subject, comprising administering to
said subject an effective amount of the composition of claim 8.
10. A method of preparing a polyclonal antibody with the
specificity of the antibody of claim 3 comprising: a) immunizing an
animal with a polypeptide of SEQ ID NO:1 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 of
SEQ ID NO:1.
11. An antibody produced by a method of claim 10.
12. A composition comprising the antibody of claim 11 and a
suitable carrier.
13. A method of making a monoclonal antibody with the specificity
of the antibody of claim 3 comprising: a) immunizing an animal with
a polypeptide of SEQ ID NO:1 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 of SEQ ID NO:1.
14. A monoclonal antibody produced by a method of claim 13.
15. A composition comprising the antibody of claim 14 and a
suitable carrier.
16. The antibody of claim 3, wherein the antibody is produced by
screening a Fab expression library.
17. The antibody of claim 3, wherein the antibody is produced by
screening a recombinant immunoglobulin library.
18. A method for detecting a polypeptide of SEQ ID NO:1 in a sample
comprising the steps of: a) incubating the antibody of claim 3 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 of SEQ ID
NO:1 in the sample.
19. A method of purifying a polypeptide of SEQ ID NO:1 from a
sample, the method comprising: a) incubating the antibody of claim
3 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 purified polypeptide of SEQ ID NO:1.
Description
[0001] This application is a divisional application of U.S.
application Ser. No. 09/388,774 filed Sep. 2, 1999, which is a
divisional application of U.S. application Ser. No. 09/074,579
filed May 7, 1998, issued Dec. 14, 1999 as U.S. Pat. No. 6,001,596,
all of which applications and patents are hereby incorporated by
reference herein.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of a growth-associated protease inhibitor heavy chain
precursor and to the use of these sequences in the diagnosis,
treatment, and prevention of reproductive, developmental,
neoplastic, and immunological disorders.
BACKGROUND OF THE INVENTION
[0003] Proteolytic processing is an essential component of normal
cell growth, differentiation, remodeling, and homeostasis. The
cleavage of peptide bonds within cells is necessary for the
maturation of precursor proteins to their active form, the removal
of signal sequences from targeted proteins, the degradation of
incorrectly folded proteins, and the controlled turnover of
peptides within the cell. Proteases participate in apoptosis,
antigen presentation, inflammation, tissue remodeling during
embryonic development, wound healing, and normal growth. They are
necessary components of bacterial, parasitic, and viral invasion
and replication within a host. Four principal categories of
mammalian proteases have been identified based on active site
structure, mechanism of action, and overall three-dimensional
structure. (Beynon, R. J. and Bond, J. S. (1994) Proteolytic
Enzymes: A Practical Approach, Oxford University Press, New York,
N.Y., pp. 1-5.)
[0004] The serine proteases (SPs) are a large family of proteolytic
enzymes that include the digestive enzymes, trypsin and
chymotrypsin; components of the complement cascade and of the
blood-clotting cascade; and enzymes that control the degradation
and turnover of macromolecules of the extracellular matrix. SPs are
so named because of the presence of a serine residue found in the
active catalytic site for protein cleavage. The active site of all
SP is composed of a triad of residues including the aforementioned
serine, an aspartate, and a histidine residue. SPs have a wide
range of substrate specificities and can be subdivided into
subfamilies on the basis of these specificities. The main
sub-families are trypases which cleave after arginine or lysine;
aspases which cleave after aspartate; chymases which cleave after
phenylalanine or leucine; metases which cleavage after methionine;
and serases which cleave after serine.
[0005] The plasma inter-.alpha.-trypsin inhibitor family molecules
are serine protease inhibitors (serpins) composed of a 240 kDa
plasma protein complex of at least five different types of
glycoproteins. These glycoproteins consist of four heavy (H) chains
and one 30 kDa light (L) chain named H1, H2, H3, H4, and L, and are
independently synthesized and proteolytically processed from
precursor proteins. (Daveau, M. et al. (1998) Arch. Biochem.
Biophys. 350:315-323; and Salier, J. P. et al. (1992) Mamm. Genome
2:233-239.) The plasma inter-.alpha.-trypsin inhibitor light chains
have sequence similarity to the Kunitz trypsin inhibitors which
appear to be present in all vertebrates (Salier, J. P. (1990)
Trends Biochem. Sci. 15:435439.) Some examples of the Kunitz
trypsin inhibitors are tissue factor pathway inhibitor, which
regulates tissue factor-induced coagulation, and protease nexin-2,
which regulates serum coagulation factor XIa. (Broze, G. J. (1995)
Annu. Rev. Med. 46:103-112; and Wagner, S. L. et al. (1993) Brain
Res. 626:90-98.) The heavy chain precursors encode a signal peptide
sequence and the mature chain. Other plasma inter-.alpha.-trypsin
inhibitor heavy chains have been described in human and rodents.
(Bourguignon, J. et al. (1993) Eur. J. Biochem. 212:771-776;
Salier, 1992, supra; and Salier, J. P. (1996) Biochem. J. 315:1-9.)
Proteases and protease inhibitory molecules may contain amino acid
sequence motifs which determine protein-protein interactions, such
as the potential metal-binding site of von Willebrand factor type
A3 (vWFA3) motif, glycine-amino acid-serine-amino acid-serine. This
motif is also required for ligand interaction in the homologous
I-type domains of integrins CR3 and LFA-1. (Huizinga, E. G. (1997)
Structure 5:1147-1156.)
[0006] The expression of the rat plasma inter-.alpha.-trypsin
inhibitor genes is regulated by inflammation in vivo. The genes are
predominantly expressed in the rat liver, but H2 and H3 mRNA is
also present in brain, intestine, and stomach. (Daveau, supra.)
[0007] Protease inhibitors play a major role in the regulation of
the activity and effect of proteases. They have been shown to
control pathogenesis in animal models of proteolytic disorders and
in the treatment of HIV. (Murphy, G. (1991) Agents Actions Suppl.
35:69-76; and Pakyz, A. and Isreal, D. (1997) J. Am. Pharm. Assoc.
(Wash.) NS37:543-551.)
[0008] The discovery of a new growth-associated protease inhibitor
heavy chain precursor and the polynucleotides encoding it satisfies
a need in the art by providing new compositions which are useful in
the diagnosis, treatment, and prevention of reproductive,
developmental, neoplastic, and immunological disorders.
SUMMARY OF THE INVENTION
[0009] The invention is based on the discovery of a new human
growth-associated protease inhibitor heavy chain precursor (GAPIP),
the polynucleotides encoding GAPIP, and the use of these
compositions for the diagnosis, treatment, or prevention of
reproductive, developmental, neoplastic, and immunological
disorders.
[0010] The invention features a substantially purified polypeptide
comprising the amino acid sequence of SEQ ID NO:1 or a fragment of
SEQ ID NO:1.
[0011] 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 a fragment of SEQ ID NO:1.
The invention also provides an isolated and purified polynucleotide
encoding the polypeptide comprising the sequence of SEQ ID NO:1 or
a fragment of SEQ ID NO:1. 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 a
fragment of SEQ ID NO:1.
[0012] 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 a fragment of SEQ ID NO:1, 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 a fragment of SEQ ID NO:1.
[0013] The invention also provides an isolated and purified
polynucleotide comprising the polynucleotide sequence of SEQ ID
NO:2 or a fragment of SEQ ID NO:2, 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 or a fragment of SEQ ID NO:2. 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 or a fragment of SEQ ID
NO:2.
[0014] The invention further provides an expression vector
comprising at least a fragment of the polynucleotide encoding the
polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of
SEQ ID NO:1. In another aspect, the expression vector is contained
within a host cell.
[0015] The invention also provides a method for producing a
polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a
fragment of SEQ ID NO:1, the method comprising the steps of: (a)
culturing the host cell comprising 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 a fragment of
SEQ ID NO:1 under conditions suitable for the expression of the
polypeptide; and (b) recovering the polypeptide from the host cell
culture.
[0016] The invention also provides a pharmaceutical composition
comprising a substantially purified polypeptide having the sequence
of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a
suitable pharmaceutical carrier.
[0017] The invention further includes a purified antibody which
binds to a polypeptide comprising the sequence of SEQ ID NO:1 or a
fragment of SEQ ID NO:1, as well as a purified agonist and a
purified antagonist of the polypeptide.
[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 a pharmaceutical composition comprising substantially
purified polypeptide having the amino acid sequence of SEQ ID NO:1
or a fragment of SEQ ID NO:1.
[0019] The invention also provides a method for treating or
preventing a developmental disorder, 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 a fragment of SEQ ID NO:1.
[0020] The invention also provides a method for treating or
preventing a neoplastic 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 a fragment of SEQ ID NO:1.
[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 a fragment of SEQ ID NO:1.
[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 a fragment of SEQ ID NO:1 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 a fragment of SEQ ID NO:1 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 a fragment of SEQ ID NO:1 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, 1F, 1G, 1H, 1I, and 1J show the
amino acid sequence (SEQ ID NO:1) and nucleic acid sequence (SEQ ID
NO:2) of GAPIP. The alignment was produced using MacDNASIS PRO
software (Hitachi Software Engineering Co. Ltd., San Bruno,
Calif.).
[0024] FIGS. 2A, 2B, 2C, 2D, 2E, 2F, and 2G show the amino acid
sequence alignments among GAPIP (688183; SEQ ID NO:1), human
pre-inter-.alpha.-trypsin inhibitor (GI 33985; SEQ ID NO:3), human
pre-inter-.alpha.-trypsin inhibitor heavy chain H1 (GI 33989; SEQ
ID NO:4), and human pre-inter-.alpha.-trypsin inhibitor heavy chain
H3 (GI 288563; SEQ ID NO:5), produced using the multisequence
alignment program of LASERGENE software (DNASTAR Inc, Madison
Wis.).
[0025] FIG. 3 shows the amino acid sequence phylogenic tree among
GAPIP (688183; SEQ ID NO:1), human pre-inter-.alpha.-trypsin
inhibitor (GI 33985; SEQ ID NO:3), human pre-inter-.alpha.-trypsin
inhibitor heavy chain H1 (GI 33989; SEQ ID NO:4), and human
pre-inter-.alpha.-trypsin inhibitor heavy chain H3 (GI 288563; SEQ
ID NO:5), produced using the multisequence alignment program of
LASERGENE software (DNASTAR Inc).
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.
[0029] 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.
[0030] Definitions
[0031] "GAPIP," as used herein, refers to the amino acid sequences
of substantially purified GAPIP 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.
[0032] The term "agonist," as used herein, refers to a molecule
which, when bound to GAPIP, increases or prolongs the duration of
the effect of GAPIP. Agonists may include proteins, nucleic acids,
carbohydrates, or any other molecules which bind to and modulate
the effect of GAPIP.
[0033] An "allelic variant," as this term is used herein, is an
alternative form of the gene encoding GAPIP. Allelic variants 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 allelic variants 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.
[0034] "Altered" nucleic acid sequences encoding GAPIP, as
described herein, include those sequences with deletions,
insertions, or substitutions of different nucleotides, resulting in
a polynucleotide the same as GAPIP or a polypeptide with at least
one functional characteristic of GAPIP. Included within this
definition are polymorphisms which may or may not be readily
detectable using a particular oligonucleotide probe of the
polynucleotide encoding GAPIP, and improper or unexpected
hybridization to allelic variants, with a locus other than the
normal chromosomal locus for the polynucleotide sequence encoding
GAPIP. 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 GAPIP. 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 GAPIP 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.
[0035] 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 GAPIP
which are preferably about 5 to about 15 amino acids in length,
most preferably 14 amino acids, and which retain some biological
activity or immunological activity of GAPIP. 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.
[0036] "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.)
[0037] The term "antagonist," as it is used herein, refers to a
molecule which, when bound to GAPIP, decreases the amount or the
duration of the effect of the biological or immunological activity
of GAPIP. Antagonists may include proteins, nucleic acids,
carbohydrates, antibodies, or any other molecules which decrease
the effect of GAPIP.
[0038] As used herein, the term "antibody" refers to intact
molecules as well as to fragments thereof, such as Fab,
F(ab').sub.2, and Fv fragments, which are capable of binding the
epitopic determinant.
[0039] Antibodies that bind GAPIP 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.
[0040] 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. 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.
[0041] The term "antisense," as used herein, refers to any
composition containing a nucleic acid sequence which is
complementary to the "sense" strand of a specific nucleic acid
sequence. 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.
[0042] 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
GAPIP, or of any oligopeptide thereof, to induce a specific immune
response in appropriate animals or cells and to bind with specific
antibodies.
[0043] 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.
[0044] 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 GAPIP or fragments of GAPIP 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.,
sodium dodecyl sulfate (SDS), and other components, e.g.,
Denhardt's solution, dry milk, salmon sperm DNA, etc.
[0045] "Consensus sequence," as used herein, refers to a nucleic
acid sequence which has been resequenced to resolve uncalled bases,
extended using XL-PCR (Applied Biosystems, Foster City Calif.) 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 Fragment Assembly system (GCG, Madison, Wis.). Some
sequences have been both extended and assembled to produce the
consensus sequence.
[0046] 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 GAPIP, by Northern analysis is indicative of the presence
of nucleic acids encoding GAPIP in a sample, and thereby correlates
with expression of the transcript from the polynucleotide encoding
GAPIP.
[0047] 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.
[0048] The term "derivative," as used herein, refers to the
chemical modification of a polypeptide sequence, or a
polynucleotide sequence. 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.
[0049] The term "similarity," as used herein, refers to a degree of
complementarity. There may be partial similarity or complete
similarity. The word "identity" may substitute for the word
"similarity." A partially complementary sequence that at least
partially inhibits an identical sequence from hybridizing to a
target nucleic acid is referred to as "substantially similar." 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 similar sequence or hybridization probe
will compete for and inhibit the binding of a completely similar
(identical) 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% similarity or
identity). In the absence of non-specific binding, the
substantially similar sequence or probe will not hybridize to the
second non-complementary target sequence.
[0050] 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 program
(DNASTAR, Inc.). The MEGALIGN 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
similarity 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.
[0051] "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.)
[0052] 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.
[0053] "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.
[0054] As used herein, the term "hybridization complex" 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).
[0055] 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.
[0056] "Immune response" can refer to conditions associated with
inflammation, 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 systemic defense
systems.
[0057] 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.
[0058] The terms "element" or "array element" as used herein in a
microarray context, refer to hybridizable polynucleotides arranged
on the surface of a substrate.
[0059] The term "modulate," as it appears herein, refers to a
change in the activity of GAPIP. For example, modulation may cause
an increase or a decrease in protein activity, binding
characteristics, or any other biological, functional, or
immunological properties of GAPIP.
[0060] The phrases "nucleic acid" or "nucleic acid sequence," as
used herein, refer to a nucleotide, oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer
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, when translated, would
produce polypeptides retaining some functional characteristic,
e.g., antigenicity, or structural domain characteristic, e.g.,
ATP-binding site, of the full-length polypeptide.
[0061] 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 translation 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.
[0062] 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.
[0063] "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 or 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.)
[0064] The term "sample," as used herein, is used in its broadest
sense. A biological sample suspected of containing nucleic acids
encoding GAPIP, or fragments thereof, or GAPIP 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.
[0065] 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.
[0066] As used herein, the term "stringent conditions" refers to
conditions which permit hybridization between polynucleotides and
the claimed polynucleotides. Stringent conditions can be defined by
salt concentration, the concentration of organic solvent (e.g.,
formamide), temperature, and other conditions 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.
[0067] For example, stringent salt concentration will ordinarily be
less than about 750 MM NaCl and 75 mM trisodium citrate, preferably
less than about 500 mM NaCl and 50 mM trisodium citrate, and most
preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
Low stringency hybridization can be obtained in the absence of
organic solvent, e.g., formamide, while high stringency
hybridization can be obtained in the presence of at least about 35%
formamide, and most preferably at least about 50% formamide.
Stringent temperature conditions will ordinarily include
temperatures of at least about 30.degree. C., more preferably of at
least about 37.degree. C., and most preferably of at least about
42.degree. C. Varying additional parameters, such as hybridization
time, the concentration of detergent, e.g., sodium dodecyl sulfate
(SDS), and the inclusion or exclusion of carrier DNA, are well
known to those skilled in the art. Various levels of stringency are
accomplished by combining these various conditions as needed. In a
preferred embodiment, hybridization will occur at 30.degree. C. in
750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more
preferred embodiment, hybridization will occur at 37.degree. C. in
500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and
100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most
preferred embodiment, hybridization will occur at 42.degree. C. in
250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and
200 .mu.g/ml ssDNA. Useful variations on these conditions will be
readily apparent to those skilled in the art.
[0068] The washing steps which follow hybridization can also vary
in stringency. Wash stringency conditions can be defined by salt
concentration and by temperature. As above, wash stringency can be
increased by decreasing salt concentration or by increasing
temperature. For example, stringent salt concentration for the wash
steps will preferably be less than about 30 mM NaCl and 3 mM
trisodium citrate, and most preferably less than about 15 mM NaCl
and 1.5 mM trisodium citrate. Stringent temperature conditions for
the wash steps will ordinarily include temperature of at least
about 25.degree. C., more preferably of at least about 42.degree.
C., and most preferably of at least about 68.degree. C. In a
preferred embodiment, wash steps will occur at 25.degree. C. in 30
mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred
embodiment, wash steps will occur at 42.degree. C. in 15 mM NaCl,
1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred
embodiment, wash steps will occur at 68.degree. C. in 15 mM NaCl,
1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on
these conditions will be readily apparent to those skilled in the
art.
[0069] 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.
[0070] A "substitution," as used herein, refers to the replacement
of one or more amino acids or nucleotides by different amino acids
or nucleotides, respectively.
[0071] "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.
[0072] A "variant" of GAPIP, 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 software.
[0073] The Invention
[0074] The invention is based on the discovery of a new human
growth-associated protease inhibitor heavy chain precursor (GAPIP),
the polynucleotides encoding GAPIP, and the use of these
compositions for the diagnosis, treatment, or prevention of
reproductive, developmental, neoplastic, and immunological
disorders.
[0075] Nucleic acids encoding the GAPIP of the present invention
were first identified in Incyte Clone 688183 from the uterus cDNA
library (UTRSNOT02) using a computer search, e.g., BLAST, 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 688183 (UTRSNOT02), 3043969 (HEAANOT01),
3112673 (BRSTNOT17), 3052595 (LNODNOTO8), 789100 (PROSTUT03),
785182 (PROSNOT05), 1505061 and 1505717 (BRAITUT07), 1794195 and
1795083 (PROSTUT05), 2125590 (BRSTNOT07), 1558218 (SPLNNOT04),
1361072 (LUNGNOT12), and 1964439 (BRSTNOT04).
[0076] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:1, as shown in
FIGS. 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, and 1J. GAPIP is 942
amino acids in length and has eight potential N-glycosylation sites
at residues N97, N127, N231, N421, N508, N776, N795, and N862;
twelve potential casein kinase II phosphorylation sites at residues
S17, S28, T112, T129, S158, S269, S354, T410, T581, S592, T676, and
S754; two potential glycosaminoglycan attachment sites at residues
S213 and S391; seventeen potential protein kinase C phosphorylation
sites at residues S55, S70, T112, S175, S182, S213, S337, S354,
T416, T458, S535, S559, T581, S611, S620, S651, and T880; one
potential tyrosine kinase phosphorylation site at residue Y919; a
potential signal peptide sequence from M1 to C14; and a vWFA3
domain, which contains the potential metal-binding site
glycine-amino acid-serine-amino acid-serine, from N295 to N440. As
shown in FIGS. 2A, 2B, 2C, 2D, 2E, 2F, and 2G, GAPIP has chemical
and structural similarity with human pre-inter-.alpha.-trypsin
inhibitor (GI 33985; SEQ ID NO:3), human pre-inter-.alpha.-trypsin
inhibitor heavy chain H1 (GI 33989; SEQ ID NO:4), and human
pre-inter-.alpha.-trypsin inhibitor heavy chain H3 (GI 288563; SEQ
ID NO:5). In particular, GAPIP and human pre-inter-.alpha.-trypsin
inhibitor share 28% identity, one potential N-glycosylation site,
four potential casein kinase II phosphorylation sites, four
potential protein kinase C phosphorylation sites, the potential
signal peptide sequence, and the vWFA3 potential metal-binding site
glycine-amino acid-serine-amino acid-serine. In addition, GAPIP and
human pre-inter-.alpha.-trypsin inhibitor heavy chains H1 and H3
share 27% and 23% identity, respectively, one potential
N-glycosylation site, four potential casein kinase II
phosphorylation sites, five potential protein kinase C
phosphorylation sites, the potential signal peptide sequence, and
the vWFA3 potential metal-binding site glycine-amino
acid-serine-amino acid-serine. As illustrated by FIG. 3, GAPIP and
human pre-inter-.alpha.-trypsin inhibitor heavy chains share a
common phylogenic heritage. A fragment of SEQ ID NO:2 from about
nucleotide 982 to about nucleotide 1011 is useful, for example, for
designing oligonucleotides or as a hybridization probe. Northern
analysis shows the expression of this sequence in various
libraries, at least 63% of which are immortalized or cancerous and
at least 26% of which involve immune response. Of particular note
is the expression of GAPIP in reproductive, gastrointestinal,
nervous, and fetal tissues.
[0077] The invention also encompasses GAPIP variants. A preferred
GAPIP 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 GAPIP amino acid sequence, and which
contains at least one functional or structural characteristic of
GAPIP.
[0078] The invention also encompasses polynucleotides which encode
GAPIP. In a particular embodiment, the invention encompasses a
polynucleotide sequence comprising the sequence of SEQ ID NO:2,
which encodes an GAPIP.
[0079] The invention also encompasses a variant of a polynucleotide
sequence encoding GAPIP. 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 GAPIP. 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. Any one of the
polynucleotide variants described above can encode an amino acid
sequence which contains at least one functional or structural
characteristic of GAPIP.
[0080] 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 GAPIP, some bearing minimal
similarity 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 GAPIP, and all such
variations are to be considered as being specifically
disclosed.
[0081] Although nucleotide sequences which encode GAPIP and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring GAPIP under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding GAPIP or its derivatives
possessing a substantially different codon usage, e.g., inclusion
of non-naturally occurring codons. 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 GAPIP 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.
[0082] The invention also encompasses production of DNA sequences
which encode GAPIP and GAPIP 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 well known in the art.
Moreover, synthetic chemistry may be used to introduce mutations
into a sequence encoding GAPIP or any fragment thereof.
[0083] 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, or a fragment of SEQ ID NO:2, under various conditions of
stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods
Enzymol. 152:399407; Kimmel, A. R. (1987) Methods Enzymol.
152:507-511.)
[0084] 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 Klenow fragment of DNA polymerase I, SEQUENASE, Taq DNA
polymerase and thermostable T7 DNA polymerase (Amersham Pharmacia
Biotech (APB), Piscataway N.J.), or combinations of polymerases and
proofreading exonucleases such as those found in the ELONGASE
amplification system (Life Technologies, Gaithersburg Md.).
Preferably, sequence preparation is automated with machines such as
the MICROLAB 2200 system (Hamilton, Reno Nev.) and the DNA ENGINE
thermal cycler (MJ Research, Watertown Mass.). Machines commonly
used for sequencing include the ABI PRISM 3700, 377 or 373 DNA
sequencing systems (Applied Biosystems), the MEGABACE 1000 DNA
sequencing system (APB), and the like.
[0085] The nucleic acid sequences encoding GAPIP may be extended
utilizing a partial nucleotide sequence and employing various
PCR-based 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 and
nested primers to amplify unknown sequence from genomic DNA within
a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.) Another method, inverse PCR, uses primers that extend
in divergent directions to amplify unknown sequence from a
circularized template. The template is derived from restriction
fragments comprising a known genomic locus and surrounding
sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res.
16:8186.) A third method, capture PCR, involves PCR amplification
of DNA fragments adjacent to known sequences 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 insert
an engineered double-stranded sequence into a region of unknown
sequence 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. 19:3055-306).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER
libraries to walk genomic DNA (Clontech, Palo Alto, Calif.). This
procedure avoids the need to screen libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, 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 template at temperatures of about
68.degree. C. to 72.degree. C.
[0086] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
In addition, random-primed libraries, which often include sequences
containing the 5' regions of genes, are 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 5'
non-transcribed regulatory regions.
[0087] 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 nucleotide-specific, laser-stimulated
fluorescent dyes, 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
and SEQUENCE NAVIGATOR, Applied Biosystems), 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 sequencing small DNA fragments which may
be present in limited amounts in a particular sample.
[0088] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode GAPIP may be cloned in
recombinant DNA molecules that direct expression of GAPIP, 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 used to express
GAPIP.
[0089] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter GAPIP-encoding sequences for a variety of purposes including,
but not limited to, modification of 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, oligonucleotide-mediated site-directed mutagenesis may
be used to introduce mutations that create new restriction sites,
alter glycosylation patterns, change codon preference, produce
splice variants, and so forth.
[0090] In another embodiment, sequences encoding GAPIP 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
Symp. Ser. 7:215-223, and Horn, T. et al. (1980) Nucl. Acids Symp.
Ser. 7:225-232.) Alternatively, GAPIP itself or a fragment thereof
may be synthesized using chemical methods. 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 GAPIP, 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.
[0091] 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, WH Freeman and Co., New York,
N.Y.)
[0092] In order to express a biologically active GAPIP, the
nucleotide sequences encoding GAPIP or derivatives thereof may be
inserted into an appropriate expression vector, i.e., a vector
which contains the necessary elements for transcriptional and
translational control of the inserted coding sequence in a suitable
host. These elements include regulatory sequences, such as
enhancers, constitutive and inducible promoters, and 5' and 3'
untranslated regions in the vector and in polynucleotide sequences
encoding GAPIP. Such elements may vary in their strength and
specificity. Specific initiation signals may also be used to
achieve more efficient translation of sequences encoding GAPIP.
Such signals include the ATG initiation codon and adjacent
sequences, e.g. the Kozak sequence. In cases where sequences
encoding GAPIP and its initiation codon and upstream regulatory
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 an in-frame ATG initiation codon should be provided by
the vector. 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 host cell system used.
(See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ.
20:125-162.)
[0093] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding GAPIP 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.)
[0094] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding GAPIP. 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 viral expression vectors (e.g.,
baculovirus); plant cell systems transformed with viral 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.
[0095] In bacterial systems, a number of cloning and expression
vectors may be selected depending upon the use intended for
polynucleotide sequences encoding GAPIP. For example, routine
cloning, subcloning, and propagation of polynucleotide sequences
encoding GAPIP can be achieved using a multifunctional E. coli
vector such as BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.)
or PSPORT 1 plasmid (Life Technologies). Ligation of sequences
encoding GAPIP into the vector's multiple cloning site disrupts the
lacZ gene, allowing a calorimetric screening procedure for
identification of transformed bacteria containing recombinant
molecules. In addition, these vectors may be useful for in vitro
transcription, dideoxy sequencing, single strand rescue with helper
phage, and creation of nested deletions in the cloned sequence.
(See, e.g., Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem.
264:5503-5509.) When large quantities of GAPIP are needed, e.g. for
the production of antibodies, vectors which direct high level
expression of GAPIP may be used. For example, vectors containing
the strong, inducible T5 or T7 bacteriophage promoter may be
used.
[0096] Yeast expression systems may be used for production of
GAPIP. A number of vectors containing constitutive or inducible
promoters, such as alpha factor, alcohol oxidase, and PGH, may be
used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In
addition, such vectors direct either the secretion or intracellular
retention of expressed proteins and enable integration of foreign
sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, supra; and Grant et al. (1987) Methods Enzymol.
153:516-54; Scorer, C. A. et al. (1994) Bio/Technology
12:181-184.)
[0097] Plant systems may also be used for expression of GAPIP.
Transcription of sequences encoding GAPIP may be driven viral
promoters, e.g., the 35S and 19S promoters of CaMV 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. (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.) In mammalian cells, a number of
viral-based expression systems may be utilized. In cases where an
adenovirus is used as an expression vector, sequences encoding
GAPIP 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 infective virus which expresses GAPIP
in 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. SV40 or
EBV-based vectors may also be used for high-level protein
expression.
[0098] Human artificial chromosomes (HACs) may also be employed to
deliver larger fragments of DNA than can be contained in and
expressed from 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.
[0099] For long term production of recombinant proteins in
mammalian systems, stable expression of GAPIP in cell lines is
preferred. For example, sequences encoding GAPIP can be transformed
into cell lines 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 a selective agent, and its presence allows
growth and recovery of cells which successfully express the
introduced sequences. Resistant clones of stably transformed cells
may be propagated using tissue culture techniques appropriate to
the cell type.
[0100] 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 and adenine
phosphoribosyltransferase genes, for use 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; neo confers resistance to the aminoglycosides
neomycin and G418; 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 and hisD, which alter cellular requirements
for metabolites. (See, e.g., Hartman, S. C. and R. C. Mulligan
(1988) Proc. Natl. Acad. Sci. 85:8047-8051.) Visible markers, e.g.,
anthocyanins, green fluorescent proteins (GFP) (Clontech, Palo
Alto, Calif.), .beta. glucuronidase and its substrate
.beta.-D-glucuronoside, or luciferase and its substrate luciferin
may 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.) 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 GAPIP is inserted within a marker
gene sequence, transformed cells containing sequences encoding
GAPIP can be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding GAPIP 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.
[0101] In general, host cells that contain the nucleic acid
sequence encoding GAPIP and that express GAPIP 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, PCR amplification, 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.
[0102] Immunological methods for detecting and measuring the
expression of GAPIP using either specific polyclonal or monoclonal
antibodies 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
GAPIP is preferred, but a competitive binding assay may be
employed. These and other assays are well known in the art. (See,
e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory
Manual, APS Press, St Paul, Minn., Section IV; Coligan, J. E. et
al. (1997 and periodic supplements) Current Protocols in
Immunology, Greene Pub. Associates and Wiley-Interscience, New
York, N.Y.; and Maddox, D. E. et al. (1983) J. Exp. Med.
158:1211-1216).
[0103] 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 GAPIP include oligolabeling, nick
translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding GAPIP, 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 Promega (Madison Wis.) or Amersham Pharmacia Biotech.
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.
[0104] Host cells transformed with nucleotide sequences encoding
GAPIP 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 retained 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 GAPIP may be designed to
contain signal sequences which direct secretion of GAPIP through a
prokaryotic or eukaryotic cell membrane.
[0105] 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 specify
protein targeting, folding, and/or activity. Different host cells
which have specific cellular machinery and characteristic
mechanisms for post-translational activities (e.g., CHO, HeLa,
MDCK, BEK293, and W138), 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.
[0106] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding GAPIP may be ligated
to a heterologous sequence resulting in translation of a fusion
protein in any of the aforementioned host systems. For example, a
chimeric GAPIP protein containing a heterologous moiety that can be
recognized by a commercially available antibody may facilitate the
screening of peptide libraries for inhibitors of GAPIP activity.
Heterologous protein and peptide moieties may also facilitate
purification of fusion proteins using commercially available
affinity matrices. Such moieties include, but are not limited to,
glutathione S-transferase (GST), maltose binding protein (MBP),
thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG,
c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their cognate fusion proteins on immobilized
glutathione, maltose, phenylarsine oxide, calmodulin, and
metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin
(HA) enable immunoaffinity purification of fusion proteins using
commercially available monoclonal and polyclonal antibodies that
specifically recognize these epitope tags. A fusion protein may
also be engineered to contain a proteolytic cleavage site located
between the GAPIP encoding sequence and the heterologous protein
sequence, so that GAPIP may be cleaved away from the heterologous
moiety following purification. Methods for fusion protein
expression and purification are discussed in Ausubel, F. M. et al.
(1995 and periodic supplements) Current Protocols in Molecular
Biology, John Wiley & Sons, New York, N.Y., ch 10. A variety of
commercially available kits may also be used to facilitate
expression and purification of fusion proteins.
[0107] In a further embodiment of the invention, synthesis of
radiolabeled GAPIP may be achieved in vitro using the TNT rabbit
reticulocyte lysate or wheat germ extract systems (Promega). These
systems couple transcription and translation of protein-coding
sequences operably associated with the T7, T3, or SP6 promoters.
Translation takes place in the presence of a radiolabeled amino
acid precursor, preferably .sup.35S-methionine.
[0108] Fragments of GAPIP 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. Various fragments of GAPIP may
be synthesized separately and then combined to produce the full
length molecule.
[0109] Therapeutics
[0110] Chemical and structural similarity exists among GAPIP and
human pre-inter-.alpha.-trypsin inhibitor (GI 33985; SEQ ID NO:3),
human pre-inter-.alpha.-trypsin inhibitor heavy chain H1 (GI 33989;
SEQ ID NO:4), and human pre-inter-.alpha.-trypsin inhibitor heavy
chain H3 (GI 288563; SEQ ID NO:5). In addition, GAPIP is expressed
in cancer, immune, reproductive, gastrointestinal, nervous, and
fetal tissues. Therefore, GAPIP appears to play a role in
reproductive, developmental, neoplastic, and immunological
disorders.
[0111] Therefore, in one embodiment, a pharmaceutical composition
comprising a substantially purified GAPIP in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a reproductive disorder. Such reproductive
disorders can include, but are 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, uterine fibroids, autoimmune disorders, ectopic
pregnancies, and teratogenesis; cancer of the breast, fibrocystic
breast disease, and 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.
[0112] In another embodiment, a vector capable of expressing GAPIP
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a reproductive disorder including, but
not limited to, those described above.
[0113] In a further embodiment, GAPIP or a fragment or derivative
thereof may be administered to a subject to treat or prevent a
reproductive disorder including, but not limited to, those provided
above.
[0114] In still another embodiment, an agonist which modulates the
activity of GAPIP may be administered to a subject to treat or
prevent a reproductive disorder including, but not limited to,
those listed above.
[0115] In one embodiment, a pharmaceutical composition comprising a
substantially purified GAPIP in conjunction with a suitable
pharmaceutical carrier may be administered to a subject to treat or
prevent a developmental disorder. The term "developmental disorder"
refers to any disorder associated with growth and differentiation,
embryogenesis, and morphogenesis involving any tissue, organ, or
system of a subject (such as the brain, adrenal gland, kidney,
skeletal or reproductive system). Such developmental disorders can
include, but are not limited to, renal tubular acidosis, anemia,
Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker
muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome
(Wilms' tumor, aniridia, genitourinary abnormalities, and mental
retardation), Smith-Magenis syndrome, myelodysplastic syndrome,
hereditary mucoepithelial dysplasia, hereditary keratodermas,
hereditary neuropathies such as Charcot-Marie-Tooth disease and
neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders
such as Syndenham's chorea and cerebral palsy, spinal bifida,
congenital glaucoma, cataract, and sensorineural hearing loss.
[0116] In another embodiment, a vector capable of expressing GAPIP
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a developmental disorder including, but
not limited to, those described above.
[0117] In a further embodiment, GAPIP or a fragment or derivative
thereof may be administered to a subject to treat or prevent a
developmental disorder including, but not limited to, those
provided above.
[0118] In still another embodiment, an agonist which modulates the
activity of GAPIP may be administered to a subject to treat or
prevent a developmental disorder including, but not limited to,
those listed above.
[0119] In one embodiment, an antagonist of GAPIP may be
administered to a subject to treat or prevent a neoplastic
disorder. Such neoplastic disorders may include, but are not
limited to, adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,
sarcoma, teratocarcinoma, and, in particular, 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, an antibody which specifically binds GAPIP 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 GAPIP.
[0120] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding GAPIP may be administered
to a subject to treat or prevent a neoplastic disorder including,
but not limited to, those described above.
[0121] In one embodiment, an antagonist of GAPIP may be
administered to a subject to treat or prevent an immunological
disorder. Such immunological disorders may include, but are not
limited to, acquired immunodeficiency syndrome (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, episodic
lymphopenia with lymphocytotoxins, erythroblastosis fetalis,
erythema nodosum, atrophic gastritis, glomerulonephritis,
Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple
sclerosis, myasthenia gravis, myocardial or pericardial
inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,
scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura,
ulcerative colitis, uveitis, Werner syndrome, complications of
cancer, hemodialysis, and extracorporeal circulation, viral,
bacterial, fungal, parasitic, protozoal, and helminthic infections,
and trauma; and arteriosclerosis, bursitis, cirrhosis, hepatitis,
mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal
nocturnal hemoglobinuria, polycythemia vera, and primary
thrombocythemia. In one aspect, an antibody which specifically
binds GAPIP 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 GAPIP.
[0122] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding GAPIP may be administered
to a subject to treat or prevent an immunological disorder
including, but not limited to, those described above. 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.
[0123] An antagonist of GAPIP may be produced using methods which
are generally known in the art. In particular, purified GAPIP may
be used to produce antibodies or to screen libraries of
pharmaceutical agents to identify those which specifically bind
GAPIP. Antibodies to GAPIP 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.
[0124] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others may be immunized by
injection with GAPIP 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.
[0125] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to GAPIP 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 GAPIP amino acids may be
fused with those of another protein, such as KLH, and antibodies to
the chimeric molecule may be produced.
[0126] Monoclonal antibodies to GAPIP 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.)
[0127] 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
GAPIP-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.)
[0128] 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.)
[0129] Antibody fragments which contain specific binding sites for
GAPIP 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.)
[0130] 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 GAPIP and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering GAPIP
epitopes is preferred, but a competitive binding assay may also be
employed. (Maddox, supra.)
[0131] In another embodiment of the invention, the polynucleotides
encoding GAPIP, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding GAPIP 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 GAPIP. Thus, complementary molecules or
fragments may be used to modulate GAPIP 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 GAPIP.
[0132] 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 to
express nucleic acid sequences complementary to the polynucleotides
encoding GAPIP. (See, e.g., Sambrook, supra; and Ausubel,
supra.)
[0133] Genes encoding GAPIP can be turned off by transforming a
cell or tissue with expression vectors which express high levels of
a polynucleotide, or fragment thereof, encoding GAPIP. 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.
[0134] 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 GAPIP. 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.
[0135] 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 GAPIP.
[0136] 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.
[0137] 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 GAPIP. 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.
[0138] 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.
[0139] 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 15:462466.) 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.
[0140] 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 GAPIP, antibodies to GAPIP, and
mimetics, agonists, antagonists, or inhibitors of GAPIP. 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.
[0141] 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.
[0142] 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.).
[0143] 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.
[0144] 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.
[0145] 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.
[0146] 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.
[0147] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks' 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.
[0148] 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.
[0149] The pharmaceutical compositions of the present invention may
be manufactured in a 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.
[0150] 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 acids. 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.
[0151] 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 GAPIP, such
labeling would include amount, frequency, and method of
administration.
[0152] 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.
[0153] 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.
[0154] A therapeutically effective dose refers to that amount of
active ingredient, for example GAPIP or fragments thereof,
antibodies of GAPIP, and agonists, antagonists or inhibitors of
GAPIP, 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 toxic to therapeutic
effects is the therapeutic index, and it can be expressed as the
LD.sub.50/ED.sub.50 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.
[0155] 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.
[0156] 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.
[0157] Diagnostics
[0158] In another embodiment, antibodies which specifically bind
GAPIP may be used for the diagnosis of disorders characterized by
expression of GAPIP, or in assays to monitor patients being treated
with GAPIP or agonists, antagonists, or inhibitors of GAPIP.
Antibodies useful for diagnostic purposes may be prepared in the
same manner as described above for therapeutics. Diagnostic assays
for GAPIP include methods which utilize the antibody and a label to
detect GAPIP 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.
[0159] A variety of protocols for measuring GAPIP, including
ELISAs, RIAs, and FACS, are known in the art and provide a basis
for diagnosing altered or abnormal levels of GAPIP expression.
Normal or standard values for GAPIP expression are established by
combining body fluids or cell extracts taken from normal mammalian
subjects, preferably human, with antibody to GAPIP under conditions
suitable for complex formation. The amount of standard complex
formation may be quantitated by various methods, preferably by
photometric means. Quantities of GAPIP expressed in subject
samples, control and disease, from biopsied tissues are compared
with the standard values. Deviation between standard and subject
values establishes the parameters for diagnosing disease.
[0160] In another embodiment of the invention, the polynucleotides
encoding GAPIP 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 GAPIP may be
correlated with disease. The diagnostic assay may be used to
determine absence, presence, and excess expression of GAPIP, and to
monitor regulation of GAPIP levels during therapeutic
intervention.
[0161] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding GAPIP or closely related molecules may be used
to identify nucleic acid sequences which encode GAPIP. 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 GAPIP, allelic variants, or related
sequences.
[0162] Probes may also be used for the detection of related
sequences, and should preferably have at least 50% sequence
identity to any of the GAPIP 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 or from genomic sequences
including promoters, enhancers, and introns of the GAPIP gene.
[0163] Means for producing specific hybridization probes for DNAs
encoding GAPIP include the cloning of polynucleotide sequences
encoding GAPIP or GAPIP 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 35S, or by enzymatic labels, such
as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and the like.
[0164] Polynucleotide sequences encoding GAPIP may be used for the
diagnosis of a disorder associated with expression of GAPIP.
Examples of such a disorder include, but are not limited to, 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, uterine
fibroids, autoimmune disorders, ectopic pregnancies, and
teratogenesis; cancer of the breast, fibrocystic breast disease,
and 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; a developmental disorder,
such as, renal tubular acidosis, anemia, Cushing's syndrome,
achondroplastic dwarfism, Duchenne and Becker muscular dystrophy,
epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor,
aniridia, genitourinary abnormalities, and mental retardation),
Smith-Magenis syndrome, myelodysplastic syndrome, hereditary
mucoepithelial dysplasia, hereditary keratodermas, hereditary
neuropathies such as Charcot-Marie-Tooth disease and
neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders
such as Syndenham's chorea and cerebral palsy, spinal bifida,
congenital glaucoma, cataract, and sensorineural hearing loss; a
neoplastic disorder, such as, adenocarcinoma, leukemia, lymphoma,
melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular,
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,
acquired immunodeficiency syndrome (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, episodic lymphopenia with
lymphocytotoxins, erythroblastosis fetalis, erythema nodosum,
atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,
gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,
irritable bowel syndrome, multiple sclerosis, myasthenia gravis,
myocardial or pericardial inflammation, osteoarthritis,
osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome,
systemic anaphylaxis, systemic lupus erythematosus, systemic
sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis,
Werner syndrome, complications of cancer, hemodialysis, and
extracorporeal circulation, viral, bacterial, fungal, parasitic,
protozoal, and helminthic infections, and trauma; and
arteriosclerosis, bursitis, cirrhosis, hepatitis, mixed connective
tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria, polycythemia vera, and primary thrombocythemia. The
polynucleotide sequences encoding GAPIP 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 GAPIP expression. Such qualitative or quantitative methods
are well known in the art.
[0165] In a particular aspect, the nucleotide sequences encoding
GAPIP may be useful in assays that detect the presence of
associated disorders, particularly those mentioned above. The
nucleotide sequences encoding GAPIP 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 GAPIP 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.
[0166] In order to provide a basis for the diagnosis of a disorder
associated with expression of GAPIP, 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 GAPIP, 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.
[0167] 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.
[0168] 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.
[0169] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding GAPIP 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 GAPIP, or a fragment of a
polynucleotide complementary to the polynucleotide encoding GAPIP,
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.
[0170] Methods which may also be used to quantitate the expression
of GAPIP 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. 212:229-236.) 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.
[0171] 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.
[0172] 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.)
[0173] In another embodiment of the invention, nucleic acid
sequences encoding GAPIP 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.)
[0174] 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 GAPIP 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.
[0175] 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., ataxia-telangiectasia 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.
[0176] In another embodiment of the invention, GAPIP, 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 GAPIP and the agent being tested may be
measured.
[0177] 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 GAPIP, or fragments thereof, and washed. Bound GAPIP is then
detected by methods well known in the art. Purified GAPIP 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.
[0178] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding GAPIP specifically compete with a test compound for binding
GAPIP. In this manner, antibodies can be used to detect the
presence of any peptide which shares one or more antigenic
determinants with GAPIP.
[0179] In additional embodiments, the nucleotide sequences which
encode GAPIP 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.
[0180] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0181] I. cDNA Library Construction
[0182] The UTRSNOT02 cDNA library was constructed from uterus
tissue obtained from a 34-year old Caucasian female (specimen
#0047A) during a vaginal hysterectomy. Pathology indicated no
diagnostic abnormality in the uterus or cervix. However, the left
ovarian tissue showed dilated follicular cystis, all embedded.
Patient history included the diagnoses of dysmenorrhea,
dyspareunia, hemorrhoids and alcohol use. The patient was not
taking any medications. Family history included malignant stomach
neoplasm in the mother; benign large bowel neoplasm in the father;
congenital heart anomaly, irritable bowel syndrome, and ulcerative
colitis in the siblings; colon cancer in the paternal aunt at age
sixty and colon cancer in paternal uncle at age eleven; and
cerebrovascular disease, type II diabetes, and depression in the
grandparents.
[0183] The frozen tissue was homogenized and lysed using a
Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Instruments,
Westbury, N.Y.) in guanidinium isothiocyanate solution. The lysate
was centrifuged over a 5.7 M CsCl cushion using an SW28 rotor in an
L8-70M ultracentrifuge (Beckman Coulter, Fullerton Calif.) for 18
hours at 25,000 rpm at ambient temperature. The RNA was extracted
with acid phenol pH 4.7, precipitated using 0.3 M sodium acetate
and 2.5 volumes of ethanol, resuspended in RNAse-free water, and
DNase treated at 37.degree. C. RNA extraction and precipitation
were repeated as before. The mRNA was then isolated using the
QIAGEN OLIGOTEX kit (QIAGEN Inc., Chatsworth Calif.) and used to
construct the cDNA library.
[0184] The mRNA was handled according to the recommended protocols
in the SUPERSCRIPT plasmid system (Life Technologies). cDNA
synthesis was initiated with a NotI-oligo d(T) primer.
[0185] Double-stranded cDNA was blunted, ligated to SalI adaptors,
digested with NotI, fractionated on a SEPHAROSE CL4B column
(Catalog #275105-01, APB), and those cDNAs exceeding 400 bp were
into the NotI and SalI sites of the PSPORT 1 vector. The plasmid
PSPORT 1 was subsequently transformed into DH5.alpha. competent
cells (Catalog #18258-012, Life Technologies).
[0186] II Isolation and Sequencing of cDNA Clones
[0187] Plasmid DNA was released from the cells and purified using
the REAL PREP 96 plasmid kit (QIAGEN Inc.). The recommended
protocol was employed except for the following changes: 1) the
bacteria were cultured in 1 ml of sterile TERRIFIC BROTH (Life
Technologies) with carbenicillin at 25 mg/l and glycerol at 0.4%;
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.
[0188] 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 the DNA ENGINE thermal
cyclers (MJ Research) and Applied Biosystems 377 DNA Sequencing
Systems; and the reading frame was determined.
[0189] III. Similarity Searching of cDNA Clones and Their Deduced
Proteins
[0190] 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 similarity 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.)
[0191] 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).
[0192] 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.-4 for peptides.
[0193] 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 similarity.
[0194] 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., BLOCKS.
BLOCKS is a weighted matrix analysis algorithm based on short amino
acid segments, or blocks, compiled from the PROSITE database.
(Bairoch, A. et al. (1997) Nucleic Acids Res. 25:217-221.) The
BLOCKS algorithm is useful for classifying genes with unknown
functions. (Henikoff S. And Henikoff G. J., Nucleic Acids Research
(1991) 19:6565-6572.) Blocks, which are 3-60 amino acids in length,
correspond to the most highly conserved regions of proteins. The
BLOCKS algorithm compares a query sequence with a weighted scoring
matrix of blocks in the BLOCKS database. Blocks in the BLOCKS
database are calibrated against protein sequences with known
functions from the SWISS-PROT database to determine the stochastic
distribution of matches. Similar databases such as PRINTS, a
protein fingerprint database, are also searchable using the BLOCKS
algorithm. (Attwood, T. K. et al. (1997) J. Chem. Inf. Comput. Sci.
37:417-424.) PRINTS is based on non-redundant sequences obtained
from sources such as SWISS-PROT, GenBank, PIR, and NRL-3D.
[0195] The BLOCKS algorithm searches for matches between a query
sequence and the BLOCKS or PRINTS database and evaluates the
statistical significance of any matches found. Matches from a
BLOCKS or PRINTS search can be evaluated on two levels, local
similarity and global similarity. The degree of local similarity is
measured by scores, and the extent of global similarity is measured
by score ranking and probability values. A score of 1000 or greater
for a BLOCKS match of highest ranking indicates that the match
falls within the 0.5 percentile level of false positives when the
matched block is calibrated against SWISS-PROT. Likewise, a
probability value of less than 1.0.times.10.sup.-3 indicates that
the match would occur by chance no more than one time in every 1000
searches. Only those matches with a cutoff score of 1000 or greater
and a cutoff probability value of 1.0.times.10.sup.-3 or less are
considered in the functional analyses of the protein sequences in
the Sequence Listing.
[0196] Nucleic and amino acid sequences of the Sequence Listing may
also be analyzed using PFAM. PFAM is a Hidden Markov Model (HMM)
based protocol useful in protein family searching. HMM is a
probabilistic approach which analyzes consensus primary structures
of gene families. (See, e.g., Eddy, S. R. (1996) Cur. Opin. Str.
Biol. 6:361-365.)
[0197] The PFAM database contains protein sequences of 527 protein
families gathered from publicly available sources, e.g., SWISS-PROT
and PROSITE. PFAM searches for well characterized protein domain
families using two high-quality alignment routines, seed alignment
and full alignment. (See, e.g., Sonnhammer, E. L. L. et al. (1997)
Proteins 28:405-420.) The seed alignment utilizes the hmmls
program, a program that searches for local matches, and a
non-redundant set of the PFAM database. The full alignment utilizes
the hmmfs program, a program that searches for multiple fragments
in long sequences, e.g., repeats and motifs, and all sequences in
the PFAM database. A result or score of 100 "bits" can signify that
it is 2.sup.100-fold more likely that the sequence is a true match
to the model or comparison sequence. Cutoff scores which range from
10 to 50 bits are generally used for individual protein families
using the SWISS-PROT sequences as model or comparison
sequences.
[0198] Two other algorithms, SIGPEPT and TM, both based on the HMM
algorithm described above (see, e.g., Eddy, supra; and Sonnhammer,
supra), identify potential signal sequences and transmembrane
domains, respectively. SIGPEPT was created using protein sequences
having signal sequence annotations derived from SWISS-PROT. It
contains about 1413 non-redundant signal sequences ranging in
length from 14 to 36 amino acid residues. TM was created similarly
using transmembrane domain annotations. It contains about 453
non-redundant transmembrane sequences encompassing 1579
transmembrane domain segments. Suitable HMM models were constructed
using the above sequences and were refined with known SWISS-PROT
signal peptide sequences or transmembrane domain sequences until a
high correlation coefficient, a measurement of the correctness of
the analysis, was obtained. Using the protein sequences from the
SWISS-PROT database as a test set, a cutoff score of 11 bits, as
determined above, correlated with 91-94% true-positives and about
4.1% false-positives, yielding a correlation coefficient of about
0.87-0.90 for SIGPEPT. A score of 11 bits for TM will typically
give the following results: 75% true positives; 1.72% false
positives; and a correlation coefficient of 0.76. Each search
evaluates the statistical significance of any matches found and
reports only those matches that score at least 11 bits.
[0199] IV. Northern Analysis
[0200] 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.)
[0201] Analogous computer techniques applying BLAST are used to
search for identical or related molecules in nucleotide databases
such as GenBank or LIFESEQ database (Incyte Genomics, Palo Alto
Calif.). 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 similar.
[0202] The basis of the search is the product score, which is
defined as: 1 % sequence identity .times. % maximum BLAST score
100
[0203] 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. Similar molecules are usually identified by
selecting those which show product scores between 15 and 40,
although lower scores may identify related molecules.
[0204] The results of Northern analysis are reported as a list of
libraries in which the transcript encoding GAPIP 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.
[0205] V. Extension of GAPIP Encoding Polynucleotides
[0206] The nucleic acid sequence of Incyte Clone 688183 was used to
design oligonucleotide primers for extending a partial nucleotide
sequence to full length. 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), 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.
[0207] Selected human cDNA libraries (Life Technologies) 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.
[0208] High fidelity amplification was obtained by following the
instructions for the XL-PCR kit (Applied Biosystems) and thoroughly
mixing the enzyme and reaction mix. PCR was performed using the DNA
ENGINE thermal cyclers (MJ Research), 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)
[0209] 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 (QIAGEN
Inc.), and trimmed of overhangs using Klenow enzyme to facilitate
religation and cloning.
[0210] 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.
[0211] 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)
[0212] 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.
[0213] In like manner, the nucleotide sequence of SEQ ID NO:2 is
used to obtain 5' regulatory sequences using the procedure above,
oligonucleotides designed for 5' extension, and an appropriate
genomic library.
[0214] VI. Labeling and Use of Individual Hybridization Probes
[0215] Hybridization probes derived from SEQ ID NO:2 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 software (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, Boston,
Mass.). The labeled oligonucleotides are substantially purified
using a SEPHADEX G-25 superfine size exclusion dextran bead column
(APB). 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: AseI, BglII, EcoRI, PstI, Xba1, or PvuII (DuPont
NEN, Boston, Mass.).
[0216] The DNA from each digest is fractionated on a 0.7% 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 film (Kodak, Rochester,
N.Y.) is exposed to the blots to film for several hours,
hybridization patterns are compared visually.
[0217] VII. Microarrays
[0218] 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.
[0219] 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. 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.
[0220] VIII. Complementary Polynucleotides
[0221] Sequences complementary to the GAPIP-encoding sequences, or
any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring GAPIP. 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
GAPIP. 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 GAPIP-encoding transcript.
[0222] IX. Expression of GAPIP
[0223] Expression and purification of GAPIP is achieved using
bacterial or virus-based expression systems. For expression of
GAPIP in bacteria, cDNA is subcloned into an appropriate vector
containing an antibiotic resistance gene and an inducible promoter
that directs high levels of cDNA transcription. Examples of such
promoters include, but are not limited to, the trp-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction
with the lac operator regulatory element. Recombinant vectors are
transformed into suitable bacterial hosts, e.g., BL21(DE3).
Antibiotic resistant bacteria express GAPIP upon induction with
isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of GAPIP
in eukaryotic cells is achieved by infecting insect or mammalian
cell lines with recombinant Autographica californica nuclear
polyhedrosis virus (AcMNPV), commonly known as baculovirus. The
nonessential polyhedrin gene of baculovirus is replaced with cDNA
encoding GAPIP by either homologous recombination or
bacterial-mediated transposition involving transfer plasmid
intermediates. Viral infectivity is maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription.
Recombinant baculovirus is used to infect Spodoptera frugiperda
(Sf9) insect cells in most cases, or human hepatocytes, in some
cases. Infection of the latter requires additional genetic
modifications to baculovirus. (See Engelhard, E. K. et al. (1994)
Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)
Hum. Gene Ther. 7:1937-1945.)
[0224] In most expression systems, GAPIP is synthesized as a fusion
protein with, e.g., glutathione S-transferase (GST) or a peptide
epitope tag, such as FLAG or 6-His, permitting rapid, single-step,
affinity-based purification of recombinant fusion protein from
crude cell lysates. GST, a 26-kilodalton enzyme from Schistosoma
japonicum, enables the purification of fusion proteins on
immobilized glutathione under conditions that maintain protein
activity and antigenicity (Pharmacia, Piscataway, N.J.). Following
purification, the GST moiety can be proteolytically cleaved from
GAPIP at specifically engineered sites. FLAG, an 8-amino acid
peptide, enables immunoaffinity purification using commercially
available monoclonal and polyclonal anti-FLAG antibodies (Eastman
Kodak, Rochester, N.Y.). 6-His, a stretch of six consecutive
histidine residues, enables purification on metal-chelate resins
(QIAGEN Inc.). Methods for protein expression and purification are
discussed in Ausubel, F. M. et al. (1995 and periodic supplements)
Current Protocols in Molecular Biology, John Wiley & Sons, New
York, N.Y., ch 10, 16. Purified GAPIP obtained by these methods can
be used directly in the following activity assay.
[0225] X. Demonstration of GAPIP Activity
[0226] Protease inhibitory activity of GAPIP is measured by the
inhibition of hydrolysis by trypsin of appropriate synthetic
peptide substrates conjugated with various chromogenic molecules in
which the degree of hydrolysis is quantitated by spectrophotometric
(or fluorometric) absorption of the released chromophore (Beynon
and Bond supra, pp.25-55). Peptide substrates are selected for
optimal activity using prepared trypsin. Chromogens commonly used
are 2-naphthylamine, 4-nitroaniline, and furylacrylic acid. Assays
are performed at ambient temperature and contain an aliquot of
trypsin, the appropriate substrate in a suitable buffer, and serial
dilutions of purified GAPIP. Reactions are carried out in an
optical cuvette and followed by the increase/decrease in absorbance
or fluorescence of the chromogen released during hydrolysis of the
peptide substrate. The baseline absorbance in the absence of GAPIP
is proportional to the trypsin activity in the assay. Reduction in
absorbance is proportional to the GAPIP activity in the assay.
[0227] XI. Functional Assays
[0228] GAPIP function is assessed by expressing the sequences
encoding GAPIP at physiologically elevated levels in mammalian cell
culture systems. cDNA is subcloned into a mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include pCMV SPORT (Life
Technologies) and pCR 3.1 (Invitrogen, Carlsbad, Calif., both of
which contain the cytomegalovirus promoter. 5-10 .mu.g of
recombinant vector are transiently transfected into a human cell
line, preferably of endothelial or hematopoietic origin, using
either liposome formulations or electroporation. 1-2 .mu.g of an
additional plasmid containing sequences encoding a marker protein
are co-transfected. Expression of a marker protein provides a means
to distinguish transfected cells from nontransfected cells and is a
reliable predictor of cDNA expression from the recombinant vector.
Marker proteins of choice include, e.g., Green Fluorescent Protein
(GFP) (Clontech, Palo Alto, Calif.), CD64, or a CD64-GFP fusion
protein. Flow cytometry (FCM), an automated, laser optics-based
technique, is used to identify transfected cells expressing GFP or
CD64-GFP, and to evaluate properties, for example, their apoptotic
state. FCM detects and quantifies the uptake of fluorescent
molecules that diagnose events preceding or coincident with cell
death. These events include changes in nuclear DNA content as
measured by staining of DNA with propidium iodide; changes in cell
size and granularity as measured by forward light scatter and 90
degree side light scatter; down-regulation of DNA synthesis as
measured by decrease in bromodeoxyuridine uptake; alterations in
expression of cell surface and intracellular proteins as measured
by reactivity with specific antibodies; and alterations in plasma
membrane composition as measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface.
Methods in flow cytometry are discussed in Ormerod, M. G. (1994)
Flow Cytometry, Oxford, New York, N.Y.
[0229] The influence of GAPIP on gene expression can be assessed
using highly purified populations of cells transfected with
sequences encoding GAPIP and either CD64 or CD64-GFP. CD64 and
CD64-GFP are expressed on the surface of transfected cells and bind
to conserved regions of human immunoglobulin G (IgG). Transfected
cells are efficiently separated from nontransfected cells using
magnetic beads coated with either human IgG or antibody against
CD64 (DYNAL, Lake Success, N.Y.). mRNA can be purified from the
cells using methods well known by those of skill in the art.
Expression of mRNA encoding GAPIP and other genes of interest can
be analyzed by Northern analysis or microarray techniques.
[0230] XII. Production of GAPIP Specific Antibodies
[0231] GAPIP substantially purified using polyacrylamide gel
electrophoresis (PAGE)(see, e.g., Harrington, M. G. (1990) Methods
Enzymol. 182:488495), or other purification techniques, is used to
immunize rabbits and to produce antibodies using standard
protocols.
[0232] Alternatively, the GAPIP amino acid sequence is analyzed
using LASERGENE software to 25 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.)
[0233] Typically, oligopeptides 15 residues in length are
synthesized using an Applied Biosystems 30 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 by, for
example, binding the peptide to plastic, blocking with 1% BSA,
reacting with rabbit antisera, washing, and reacting with
radio-iodinated goat anti-rabbit IgG.
[0234] XIII. Purification of Naturally Occurring GAPIP Using
Specific Antibodies
[0235] Naturally occurring or recombinant GAPIP is substantially
purified by immunoaffinity chromatography using antibodies specific
for GAPIP. An immunoaffinity column is constructed by covalently
coupling anti-GAPIP antibody to an activated chromatographic resin,
such as CNBr-activated SEPHAROSE (APB). After the coupling, the
resin is blocked and washed according to the manufacturer's
instructions.
[0236] Media containing GAPIP are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of GAPIP (e.g., high ionic strength buffers
in the presence of detergent). The column is eluted under
conditions that disrupt antibody/GAPIP 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 GAPIP is collected.
[0237] XIV. Identification of Molecules Which Interact with
GAPIP
[0238] GAPIP, or biologically active fragments thereof, are labeled
with 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 GAPIP,
washed, and any wells with labeled GAPIP complex are assayed. Data
obtained using different concentrations of GAPIP are used to
calculate values for the number, affinity, and association of GAPIP
with the candidate molecules.
[0239] 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
* * * * *