CA2333006A1 - Calmodulin related protein - Google Patents
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- CA2333006A1 CA2333006A1 CA002333006A CA2333006A CA2333006A1 CA 2333006 A1 CA2333006 A1 CA 2333006A1 CA 002333006 A CA002333006 A CA 002333006A CA 2333006 A CA2333006 A CA 2333006A CA 2333006 A1 CA2333006 A1 CA 2333006A1
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- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4728—Calcium binding proteins, e.g. calmodulin
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
The invention provides a human calmodulin related protein (CALREP) and polynucleotides which identify and encode CALREP. The invention also provide s expression vectors, host cells, antibodies, agonists, and antagonist. The invention also provides methods for diagnosing, treating or preventing disorders associated with expression of CALREP.
Description
WO 99/61609 PCTNS99/10?47 CALMODULIN RELATED PROTEIN
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of a calmodulin-like protein and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative and neurological disorders.
BACKGROUND OF THE INVENTION
t o In nearly all eukaryotic cells, calcium {Ca'-') functions as an intracellular signaling molecule in diverse cellular processes including cell proliferation, neurotransmitter secretion. glycogen metabolism, and skeletal muscle contraction. Within a resting cell.
the concentration of Ca'-' in the cytosol is extremely low, <10-' M. However, when the cell is stimulated by an external signal. such as a neural impulse or a growth factor, the ~5 cytosolic concentration of Ca?' increases by about SO-fold. This influx of Ca-'- is caused by the opening of plasma membrane Ca'-y channels and by the release of Ca'-+
from intracellular stores such as the endoplasmic reticulum. Ca'-' directly activates regulatory enzymes, such as protein kinase C, which trigger signal transduction pathways.
Ca'-+ also binds to specific Ca'-'-binding proteins. such as calmodulin, which then activate multiple 3o target proteins including enzymes, membrane transport pumps, and ion channels.
Many Ca'-'-binding proteins are characterized by the presence of one or more EF-hand Ca'-'-binding motifs. (Reviewed in Cello, M. R. et al. (1996) Guidebook to the Calcium-Binding Proteins, Oxford University Press, New York, NY, pp. I S-20, 34-43, and 152-1 S5.) The EF-hand motif is comprised of 12 amino acids flanked by a-helices.
35 Acidic residues are generally conserved at positions l, 3, 5, 7, 9, and 12, and glycine is often conserved at position 6. At least 250 EF-hand proteins defining 39 subfamilies have been identified in a variety of tissues. For example, calmodulin is a ubiquitous, highly conserved EF-hand protein that regulates cell proliferation and cell cycle progression.
Troponin C is a muscle-specific calmodulin-like protein involved in actin-mediated 3o muscle contraction. S 100(3 is an EF-hand protein abundantly expressed in the nervous system, particularly in glial cells. Recently, a messenger RNA (mRNA) encoding a calmodulin-related protein called NB-1 has been isolated from normal human mammary epithelial cells (HMECs). (Yaswen, P. et al. (1990) Proc. Natl. Acad. Sci. USA
87:7360-7364.) NB-1 contains four EF-hands and shares 85% identity with calmoduiin at the amino acid level. NB-I mRNA expression is decreased greater than SO-fold in HMECs s tumorigenically transformed in vitro. In addition, NB-1 transcripts are detected in normal breast, prostate. cervix, and epidermal tissues, but not in immortalized cell lines derived from tumors of these tissues. These observations suggest that NB-1 expression is associated with tumor suppression.
Aberrant expression of EF-hand Ca'-' binding proteins has been implicated in the to progression and diagnosis of certain disease states. (Cello, supra.) For example, calmodulin levels, in contrast with NB-1 levels, are increased several-fold in tumor cells.
Likewise, the levels of S 1 OO~i are increased in the blood and cerebrospinal fluid of patients with neurological injury resulting from cerebral infarction, transient ischemic attacks, hemorrhagia, head trauma, and Down"s syndrome. Furthermore, 5100(i and other neural-1 s specific EF-hand proteins may also protect against neurodegenerative disorders, such as Alzheimer's, Parkinson's, and Huntington's diseases. In addition, antibodies against S I OO~i are used to diagnose and classify tumors of glial cell origin.
The discovery of a new calmodulin related protein and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in 20 the diagnosis, treatment, and prevention of cell proliferative and neurological disorders.
SUMMARY OF THE INVENTION
The invention is based on the discovery of a new human calmoduiin related protein (CALREP), the polynucleotides encoding CALREP, and the use of these compositions for 2s the diagnosis, treatment, or prevention of cell proliferative and neurological disorders.
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.
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 3o 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.
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.
to The invention also provides an isolated and purified polynucleotide comprising the polynucleotide sequence of SEQ ID N0:2 or a fragment of SEQ ID N0:2, and an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide comprising the polynucleotide sequence of SEQ ID N0:2 or a fragment of SEQ ID N0:2. The invention also provides an isolated and purified ~ 5 polynucleotide having a sequence complementary to the polynucleotide comprising the polynucleotide sequence of SEQ ID N0:2 or a fragment of SEQ ID N0:2.
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 2o within a host cell.
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 25 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.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the sequence of SEQ ID NO:1 or a fragment of 3o SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
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.
The invention also provides a method for treating or preventing a cell proliferative disorder associated with decreased expression of CALREP, 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.
The invention also provides a method for treating or preventing a cell proliferative disorder associated with increased expression of CALREP, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist to of the polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ
ID NO:1.
The invention also provides a method for treating or preventing a neurological disorder associated with decreased expression of CALREP, 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: I or a fragment of SEQ ID NO: I .
The invention also provides a method for treating or preventing a neurological disorder associated with increased expression of CALREP, 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.
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: I 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: I 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 3o 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 polymerise chain reaction prior to the hybridizing step.
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of a calmodulin-like protein and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative and neurological disorders.
BACKGROUND OF THE INVENTION
t o In nearly all eukaryotic cells, calcium {Ca'-') functions as an intracellular signaling molecule in diverse cellular processes including cell proliferation, neurotransmitter secretion. glycogen metabolism, and skeletal muscle contraction. Within a resting cell.
the concentration of Ca'-' in the cytosol is extremely low, <10-' M. However, when the cell is stimulated by an external signal. such as a neural impulse or a growth factor, the ~5 cytosolic concentration of Ca?' increases by about SO-fold. This influx of Ca-'- is caused by the opening of plasma membrane Ca'-y channels and by the release of Ca'-+
from intracellular stores such as the endoplasmic reticulum. Ca'-' directly activates regulatory enzymes, such as protein kinase C, which trigger signal transduction pathways.
Ca'-+ also binds to specific Ca'-'-binding proteins. such as calmodulin, which then activate multiple 3o target proteins including enzymes, membrane transport pumps, and ion channels.
Many Ca'-'-binding proteins are characterized by the presence of one or more EF-hand Ca'-'-binding motifs. (Reviewed in Cello, M. R. et al. (1996) Guidebook to the Calcium-Binding Proteins, Oxford University Press, New York, NY, pp. I S-20, 34-43, and 152-1 S5.) The EF-hand motif is comprised of 12 amino acids flanked by a-helices.
35 Acidic residues are generally conserved at positions l, 3, 5, 7, 9, and 12, and glycine is often conserved at position 6. At least 250 EF-hand proteins defining 39 subfamilies have been identified in a variety of tissues. For example, calmodulin is a ubiquitous, highly conserved EF-hand protein that regulates cell proliferation and cell cycle progression.
Troponin C is a muscle-specific calmodulin-like protein involved in actin-mediated 3o muscle contraction. S 100(3 is an EF-hand protein abundantly expressed in the nervous system, particularly in glial cells. Recently, a messenger RNA (mRNA) encoding a calmodulin-related protein called NB-1 has been isolated from normal human mammary epithelial cells (HMECs). (Yaswen, P. et al. (1990) Proc. Natl. Acad. Sci. USA
87:7360-7364.) NB-1 contains four EF-hands and shares 85% identity with calmoduiin at the amino acid level. NB-I mRNA expression is decreased greater than SO-fold in HMECs s tumorigenically transformed in vitro. In addition, NB-1 transcripts are detected in normal breast, prostate. cervix, and epidermal tissues, but not in immortalized cell lines derived from tumors of these tissues. These observations suggest that NB-1 expression is associated with tumor suppression.
Aberrant expression of EF-hand Ca'-' binding proteins has been implicated in the to progression and diagnosis of certain disease states. (Cello, supra.) For example, calmodulin levels, in contrast with NB-1 levels, are increased several-fold in tumor cells.
Likewise, the levels of S 1 OO~i are increased in the blood and cerebrospinal fluid of patients with neurological injury resulting from cerebral infarction, transient ischemic attacks, hemorrhagia, head trauma, and Down"s syndrome. Furthermore, 5100(i and other neural-1 s specific EF-hand proteins may also protect against neurodegenerative disorders, such as Alzheimer's, Parkinson's, and Huntington's diseases. In addition, antibodies against S I OO~i are used to diagnose and classify tumors of glial cell origin.
The discovery of a new calmodulin related protein and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in 20 the diagnosis, treatment, and prevention of cell proliferative and neurological disorders.
SUMMARY OF THE INVENTION
The invention is based on the discovery of a new human calmoduiin related protein (CALREP), the polynucleotides encoding CALREP, and the use of these compositions for 2s the diagnosis, treatment, or prevention of cell proliferative and neurological disorders.
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.
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 3o 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.
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.
to The invention also provides an isolated and purified polynucleotide comprising the polynucleotide sequence of SEQ ID N0:2 or a fragment of SEQ ID N0:2, and an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide comprising the polynucleotide sequence of SEQ ID N0:2 or a fragment of SEQ ID N0:2. The invention also provides an isolated and purified ~ 5 polynucleotide having a sequence complementary to the polynucleotide comprising the polynucleotide sequence of SEQ ID N0:2 or a fragment of SEQ ID N0:2.
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 2o within a host cell.
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 25 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.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the sequence of SEQ ID NO:1 or a fragment of 3o SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
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.
The invention also provides a method for treating or preventing a cell proliferative disorder associated with decreased expression of CALREP, 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.
The invention also provides a method for treating or preventing a cell proliferative disorder associated with increased expression of CALREP, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist to of the polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ
ID NO:1.
The invention also provides a method for treating or preventing a neurological disorder associated with decreased expression of CALREP, 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: I or a fragment of SEQ ID NO: I .
The invention also provides a method for treating or preventing a neurological disorder associated with increased expression of CALREP, 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.
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: I 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: I 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 3o 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 polymerise chain reaction prior to the hybridizing step.
BRIEF DESCRIPTION OF THE FIGURES
Figures 1 A, 1 B, 1 C, and 1 D show the amino acid sequence (SEQ ID NO:1 ) and nucleic acid sequence (SEQ ID N0:2) of CALREP. The alignment was produced using MacDNASIS PROTM software (Hitachi Software Engineering Co. Ltd., San Bruno, CA).
Figure 2 shows the amino acid sequence alignment between the EF-hand domains of CALREP (2109176; SEQ ID NO:I ) and human NB-1 (GI 189081; SEQ ID N0:3), produced using the multisequence alignment program of LASERGENETM software (DNASTAR Inc, Madison WI).
to DESCRIPTION OF THE INVENTION
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 ~ 5 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.
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.
2o 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.
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 2s invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, vectors, and methodologies which are reported in the publications and which might be used in 30 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.
Figures 1 A, 1 B, 1 C, and 1 D show the amino acid sequence (SEQ ID NO:1 ) and nucleic acid sequence (SEQ ID N0:2) of CALREP. The alignment was produced using MacDNASIS PROTM software (Hitachi Software Engineering Co. Ltd., San Bruno, CA).
Figure 2 shows the amino acid sequence alignment between the EF-hand domains of CALREP (2109176; SEQ ID NO:I ) and human NB-1 (GI 189081; SEQ ID N0:3), produced using the multisequence alignment program of LASERGENETM software (DNASTAR Inc, Madison WI).
to DESCRIPTION OF THE INVENTION
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 ~ 5 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.
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.
2o 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.
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 2s invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, vectors, and methodologies which are reported in the publications and which might be used in 30 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.
"CALREP," as used herein, refers to the amino acid sequences of substantially purified CALREP 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.
The term "agonist," as used herein, refers to a molecule which, when bound to CALREP, increases or prolongs the duration of the effect of CALREP. Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of CALREP.
to An "allelic variant," as this term is used herein, is an alternative form of the gene encoding CALREP. 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 ~ 5 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.
"Altered" nucleic acid sequences encoding CALREP, as described herein, include those sequences with deletions, insertions, or substitutions of different nucleotides, 2o resulting in a polynucleotide the same as CALREP or a polypeptide with at least one functional characteristic of CALREP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding CALREP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide 25 sequence encoding CALREP. 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 CALREP. 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 3o the biological or immunological activity of CALREP 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.
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 CALREP which are preferably about 5 to about I S amino acids in length, most preferably 14 amino acids, and which retain some biological activity or immunological activity of CALREP. Where "amino t o 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.
"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, NY, pp.l-5.) The term "antagonist," as it is used herein, refers to a molecule which, when bound 2o to CALREP, decreases the amount or the duration of the effect of the biological or immunological activity of CALREP. Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules which decrease the effect of CALREP.
As used herein, the term "antibody" refers to intact molecules as well as to fragments thereof, such as Fab, F(ab'),, and Fv fragments, which are capable of binding the epitopic determinant. Antibodies that bind CALREP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers 3o that are chemically coupled to peptides include bovine serum albumin, thyroglobuiin, 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.
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.
i 5 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 CALREP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
2o The terms "complementary" or ''complementarity," as used herein, refer to the natural binding of polynucleotides 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 25 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.
3o 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 _8_ composition may comprise a dry formulation, an aqueous solution, or a sterile composition. Compositions comprising polynucleotide sequences encoding CALREP
or fragments of CALREP 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 s carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts, e.g., NaCI, detergents, e.g.,sodium dodecyl sulfate (SDS), and other components, e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
"Consensus sequence," as used herein, refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, extended using XL-PCRTM (Perkin Elmer, Norwalk, CT) 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 GELVIEWTM Fragment Assembly system (GCG, Madison, WI). Some sequences have been both extended and assembled to produce the consensus sequence.
~ 5 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 CALREP, by Northern analysis is indicative of the presence of nucleic acids encoding CALREP in a sample, and thereby correlates with expression of the transcript from the polynucleotide encoding CALREP.
20 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.
The term "derivative," as used herein, refers to the chemical modification of a polypeptide sequence, or a polynucleotide sequence. Chemical modifications of a 25 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 3o was derived.
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 t o 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.
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 MegAlignTM program (DNASTAR, Inc., Madison WI). The MegAlignT"' 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.
183:62b-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions.
"Human artificial chromosomes" (HACs), as described herein, are linear -lo-microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain ali 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.) 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.
"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.
to 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., Cot or Rot 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).
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.
"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.
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.
The terms "element" or "array element" as used herein in a microarray context, refer to hybridizable polynucleotides arranged on the surface of a substrate.
The term "modulate," as it appears herein, refers to a change in the activity of CALREP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of CALREP.
-il-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.
The terms "operably associated" or "operably linked," as used herein, refer to 1o 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 t5 operator sequences that control expression of the polypeptide.
The term "oligonucleotide," as used herein, refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 1 ~ 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 ?o substantially equivalent to the terms "amplimer," "primer." "oligomer," and "probe." as these terms are commonly defined in the art.
"Peptide nucleic acid" (PNA), as used herein, refers to an antisense molecule or anti-gene agent which comprises an oiigonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine.
The terming!
25 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.) The term "sample," as used herein, is used in its broadest sense. A biological 3o sample suspected of containing nucleic acids encoding CALREP, or fragments thereof, or CALREP 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.
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.
As used herein, the term "stringent conditions" refers to conditions which permit to 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.
~ 5 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.
A "substitution," as used herein, refers to the replacement of one or more amino 2o acids or nucleotides by different amino acids or nucleotides, respectively.
"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 25 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 3o chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
A ''variant" of CALREP. 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, LASERGENETM software.
THE INVENTION
The invention is based on the discovery of a new human calmodulin related protein (CALREP), the polynucleotides encoding CALREP, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative and neurological disorders.
Nucleic acids encoding the CALREP of the present invention were first identified in Incyte Clone 2109176 from the brain tumor cDNA library (BRAITUT03) using a computer search, e.g., BLAST, for amino acid sequence alignments. A consensus sequence, SEQ ID N0:2, was derived from the following overlapping and/or extended nucleic acid sequences: Incyte Clones 2109176 (BRAITUT03), 2653689 (THYMNOT04), 2878785 (UTRSTUTOS), and 959509 (BRSTTUT03).
2o In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:1, as shown in Figures 1 A. 1 B, 1 C, and 1 D.
CALREP is 386 amino acids in length and has eight potential casein kinase II
phosphorylation sites at 562, T71, S116, S196, S223. T296, S320, and 5369, and five potential protein kinase C phosphorylation sites at T71, T97, 5116, T296, and 5320. In 35 addition, both BLOCKS and PFAM analyses reveal an EF-hand motif within the region from about N324 to L352. Positions 1, 3, S, 7, and 9 of the EF-hand consensus motif are conserved in CALREP at D333, E335, S337, Y339, and D341. The conserved glycine at position 6 of the EF-hand consensus motif is also present in CALREP at 6338.
Secondary structure analysis also indicates that the regions flanking the putative EF-hand are 3o predicted to form a-helices. As shown in Figure 2, the EF-hand region of CALREP has chemical and structural similarity with the region comprising the third EF-hand of NB-1 (GI 189081; SEQ ID N0:3). Note that in Figure 2, only the relevant EF-hand regions of WO 99/61b09 PCT/US99/10747 CALREP and NB-1 are shown, and the amino acid residues are numbered based on their positions in the complete protein sequences as reported in the Sequence Listing. CALREP
and NB-1 share 30% identity within the regions shown. In addition, the potential phosphorylation sites at S196 and S369 of CALREP are conserved in NB-1. A
region of unique sequence in CALREP from about amino acid 21 to about amino acid 30 is encoded by a fragment of SEQ ID N0:2 from about nucleotide 149 to about nucleotide 178.
Northern analysis shows the expression of this sequence in various libraries, at least 67%
of which are associated with cancerous or proliferating tissue and at least 39% of which are associated with the immune response. In addition, 22% of the libraries expressing t o CALREP are derived from reproductive tissue, and 22% are derived from neurological tissue, particularly Alzheimer's and tumor-associated brain tissue.
The invention also encompasses CALREP variants. A preferred CALR.EP 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 CALREP amino acid ~ 5 sequence, and which contains at least one functional or structural characteristic of CALREP.
The invention also encompasses polynucleotides which encode CALREP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising the sequence of SEQ ID N0:2, which encodes an CALREP.
2o The invention also encompasses a variant of a polynucleotide sequence encoding CALREP. 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 CALREP. A
particular aspect of the invention encompasses a variant of SEQ ID N0:2 which has at 25 least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to SEQ ID N0:2. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of CALREP.
It will be appreciated by those skilled in the art that as a result of the degeneracy of 3o the genetic code. a multitude of polynucieotide sequences encoding CALREP, 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 CALREP, and all such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode CALREP and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring CALREP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding CALREP or its derivatives possessing a ~o 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 CALREP and its derivatives without altering the encoded t 5 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.
The invention also encompasses production of DNA sequences which encode CALREP and CALREP derivatives, or fragments thereof, entirely by synthetic chemistry.
3o 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 CALREP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are capable 25 of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID N0:2, or a fragment of SEQ ID N0:2, under various conditions of stringency.
(See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407;
Kimmel, A.R. (1987) Methods Enzymol. 152:507-511.) For example, stringent salt concentration will ordinarily be less than about 750 mM NaCI and 75 mM trisodium citrate, preferably 30 less than about 500 mM NaCI and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCI 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°C, more preferably of at least about 37°C, and most preferably of at least about 42°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°C in 750 mM NaCI, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37°C in S00 mM NaCI, 50 mM trisodium citrate, 1% SDS, 35%
formamide, and 100 ~cg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42°C in 250 mM NaCI, 25 mM trisodium citrate, 1% SDS, 50 formamide, and 200 ~cg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
~5 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 NaCI and 3 mM trisodium citrate, and most preferably less than zo about 15 mM NaCI and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include temperature of at least about 25°C, more preferably of at least about 42°C, and most preferably of at least about 68°C. In a preferred embodiment, wash steps will occur at 25°C in 30 mM NaCI, 3 mM trisodium citrate. and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42°C
in 15 mM
z5 NaCI, 1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C in 15 mM NaCI, 1.5 mM trisodium citrate, and 0.1% SDS.
Additional variations on these conditions will be readily apparent to those skilled in the art.
Methods for DNA sequencing are well known and generally available in the art 3o 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~ (US
Biochemical Corp., Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable -m-polymerase (Amersham,. Chicago, IL), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASETM Amplification System (Gteco BRL, Gaithersburg, MD). Preferably, the process is automated with machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), Peltier Thermal Cycler (PTC200;
MJ
Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
The nucleic acid sequences encoding CALREP 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 t o 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 t5 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 at. (1991) PCR Methods Applic. 1:111-119.) In this method. multiple restriction enzyme digestions and ligations may be used to insert an 2o 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 PromoterFinderTM libraries to walk genomic DNA (Clontech, Palo Alto, CA). This procedure avoids the need to screen 25 libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGOTM
4.06 Primer Analysis software (National Biosciences Inc., Plymouth, MN) 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°C to 72°C.
3o 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.
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., GenotyperT"' and Sequence NavigatorTM, Perkin Elmer), and the entire process from ~ o 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.
In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode CALREP may be cloned in recombinant DNA molecules that direct ~ 5 expression of CALREP, 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 CALREP.
The nucleotide sequences of the present invention can be engineered using 2o methods generally known in the art in order to alter CALREP-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 25 may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
In another embodiment, sequences encoding CALREP may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al. ( 1980) Nucl. Acids Res. Symp. Ser. 215-223, and Horn, T. et al. ( 1980) Nucl.
3o Acids Res. Symp. Ser. 225-232.) Alternatively, CALREP 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 431 A Peptide Synthesizer (Perkin Elmer). Additionally, the amino acid sequence of CALREP, 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.
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, NY.) In order to express a biologically active CALREP, the nucleotide sequences encoding CALREP 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 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding CALREP. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding CALREP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding CALREP and its initiation codon 20 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 25 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.) Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding CALREP and appropriate 3o 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, NY, ch. 4, 8, and 16-17; and Ausubel. F.M. et al. (1995, and periodic supplements) Current Protocols in Molecular BioloQV, John Wiley &
Sons, New York, NY, ch. 9, 13, and 16.) A variety of expression vector/host systems may be utilized to contain and express sequences encoding CALREP. 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 t0 vines (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.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding CALREP.
For example, routine cloning, subcloning, and propagation of polynucleotide sequences ~ 5 encoding CALREP can be achieved using a multifunctional E. coli vector such as Bluescript~ (Stratagene) or pSportlTM plasmid (Gt6co BRL). Legation of sequences encoding CALREP into the vector's multiple cloning site disrupts the lacZ
gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition. these vectors may be useful for in vitro transcription, 2o 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 CALREP are needed, e.g.
for the production of antibodies, vectors which direct high level expression of CALREP
may be used. For example, vectors containing the strong, inducible T5 or T7 bacteriophage 25 promoter may be used.
Yeast expression systems may be used for production of CALREP. 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~astoris.
In addition, such vectors direct either the secretion or intracellular retention of expressed 3o 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.) Plant systems may also be used for expression of CALREP. Transcription of sequences encoding CALREP 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 aI. (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 to Yearbook of Science and Technolo~y (1992) McGraw Hill, New York, NY; 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 CALREP may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E 1 or E3 region of the viral genome may be used to obtain infective virus which expresses CALREP 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.
2o 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.
For long term production of recombinant proteins in mammalian systems, stable expression of CALREP in cell lines is preferred. For example, sequences encoding CALREP 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 3o 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.
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- or apr 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 G-418; and als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F. et al {1981) J. Mol.
Biol. 150:1-14; and Murry, s_unra.) 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, CA),13 glucuronidase and its substrate 13-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.
X5:121-131.) Although the presence/absence of marker gene expression suggests that the gene of ?o interest is also present, the presence and expression of the gene may need to be confirmed.
For example, if the sequence encoding CALREP is inserted within a marker gene sequence, transformed cells containing sequences encoding CALREP can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding CALREP 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.
In general, host cells that contain the nucleic acid sequence encoding CALREP
and that express CALREP 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
3o 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.
WO 99/61609 PCTNS99/10?47 Immunological methods for detecting and measuring the expression of CALREP
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 CALREP 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, MN, Section IV;
Coligan, J. E. et al. ( 1997 and periodic supplements) Current Protocols in Immunolo~y, to Greene Pub. Associates and Wiley-Interscience, New York, NY; and Maddox, D.E. et al.
(1983) J. Exp. Med. 158:1211-1216).
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 CALREP include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding CALREP, 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 2o such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Pharmacia & Upjohn (Kalamazoo, MI), Promega (Madison, WI), and U.S. Biochemical Corp. (Cleveland, OH).
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.
Host cells transformed with nucleotide sequences encoding CALREP 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 CALREP may be designed to contain signal sequences which direct secretion of CALREP
through a prokaryotic or eukaryotic cell membrane.
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, HEK293, and WI38), are available from the American Type Culture Collection (ATCC, Bethesda, MD) and may be chosen to ensure the correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding CALREP 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 CALREP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of CALREP 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, 2o 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 CALREP encoding sequence and the heterologous protein sequence, so that CALREP 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 3o Molecular Biolo~y, John Wiley & Sons, New York, NY, ch 10. A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
In a further embodiment of the invention. synthesis of radiolabeled CALREP may be achieved in vitro using the TNTTM rabbit reticulocyte lysate or wheat germ extract systems (Promega, Madison, WI). 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 3sS-methionine.
Fragments of CALREP 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 Peptide Synthesizer (Perkin Elmer). Various fragments of CALREP may be synthesized separately and then combined to produce the full length molecule.
THERAPEUTICS
Chemical and structural similarity exists between CALREP and the EF-hand domain, particularly that of NB-1 from human (GI 189081). In addition, CALREP
is expressed in proliferating and neurological tissue. Therefore, CALREP appears to play a role in cell proliferative and neurological disorders.
Therefore, in one embodiment, CALREP or a fragment or derivative thereof may ?o be administered to a subject to treat or prevent a cell proliferative disorder associated with decreased expression of CALREP. Such disorders can include, but are not limited to, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia; cancers including adenocarcinoma, 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 immune disorders such as leukemia, lymphoma, rheumatoid arthritis, dermatitis, inflammation, systemic lupus erythematosus and other autoimmune diseases.
In another embodiment, a vector capable of expressing CALREP or a fragment or derivative thereof may be administered to a subject to treat or prevent a cell proliferative disorder including, but not limited to, those described above.
In a further embodiment, a pharmaceutical composition comprising a substantially purified CALREP in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a cell proliferative disorder including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of CALREP
may be administered to a subject to treat or prevent a cell proliferative disorder including, but not limited to, those listed above.
In a further embodiment, an antagonist of CALREP may be administered to a subject to treat or prevent a cell proliferative disorder associated with increased expression of CALREP. Such disorders can include, but are not limited to, those discussed above. In one aspect. an antibody which specifically binds CALREP 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 CALREP.
t 5 In an additional embodiment, a vector expressing the complement of the polynucleotide encoding CALREP may be administered to a subject to treat or prevent a cell proliferative disorder including, but not limited to, those described above.
In another embodiment, CALREP or a fragment or derivative thereof may be administered to a subject to treat or prevent a neurological disorder associated with 30 decreased expression of CALREP. Such disorders can include. but are not limited to, akathesia, Alzheimer's disease, amnesia, amyotrophic lateral sclerosis, bipolar disorder, catatonia, cerebral neoplasms, cerebral infarctions, dementia, depression, diabetic neuropathy, Down's syndrome, tardive dyskinesia, dystonias, epilepsy, head trauma, Huntington's disease, peripheral neuropathy, multiple sclerosis, neurofibromatosis, 25 Parkinson's disease, paranoid psychoses, postherpetic neuralgia, schizophrenia, and Tourette's disorder.
In another embodiment, a vector capable of expressing CALREP or a fragment or derivative thereof may be administered to a subject to treat or prevent a neurological disorder including, but not limited to, those described above.
3o In a further embodiment, a pharmaceutical composition comprising a substantially purified CALREP in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a neurological disorder including, but not _2~_ limited to. those provided above.
In still another embodiment, an agonist which modulates the activity of CALREP
may be administered to a subject to treat or prevent a neurological disorder including, but not limited to, those listed above.
In a further embodiment, an antagonist of CALREP may be administered to a subject to treat or prevent a neurological disorder associated with increased expression of CALREP. Such disorders can include, but are not limited to, those discussed above. In one aspect, an antibody which specifically binds CALREP may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical I o agent to cells or tissue which express CALREP.
In an additional embodiment. a vector expressing the complement of the polynucleotide encoding CALREP may be administered to a subject to treat or prevent a neurological disorder including, but not limited to, those described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists, I S 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 2o 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.
An antagonist of CALREP may be produced using methods which are generally known in the art. In particular, purified CALREP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind 25 CALREP. Antibodies to CALREP 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.
30 For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans. and others may be immunized by injection with CALREP 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.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to CALREP 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 to 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 CALREP amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
Monoclonal antibodies to CALREP 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.) 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 CALREP-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.) 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.) Antibody fragments which contain specific binding sites for CALREP 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 !o 246:1275-1281.) 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 !5 formation between CALREP and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering CALREP
epitopes is preferred, but a competitive binding assay may also be employed.
(Maddox, supra. ) In another embodiment of the invention, the polynucleotides encoding CALREP, 20 or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect. the complement of the polynucleotide encoding CALREP 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 CALREP. Thus, complementary molecules or fragments may be used to 25 modulate CALREP 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 CALREP.
Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia 3o 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 CALREP. (See, e.g., Sambrook, suyra; and Ausubel, s, unra.) Genes encoding CALREP can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide, or fragment thereof, encoding CALREP. 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 i o system.
As mentioned above, modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA. RNA, or PNA) to the control, S', or regulatory regions of the gene encoding CALREP.
Oligonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +10 from the t5 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, ?o B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt.
Kisco. NY, pp. I63-177.) A complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific 25 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 endonucieolytic cleavage of sequences encoding CALREP.
3o 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 WO 99/61b09 PCT/US99/10747 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.
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 chemic2l synthesis. Alternatively, RNA molecules may be generated by ! o in vitro and in vivo transcription of DNA sequences encoding CALREP. 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.
!5 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 2o 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.
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 25 introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al. ( 1997) Nature Biotechnology 15:462-466.) Any of the therapeutic methods described above may be applied to any subject in 3o need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
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 CALREP, antibodies to CALREP, and mimetics, agonists, antagonists, or inhibitors of CALREP. 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.
The pharmaceutical compositions utilized in this invention may be administered by t o 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.
In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries ~ 5 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 Remin~ton's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).
Pharmaceutical compositions for oral administration can be formulated using 2o 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.
Pharmaceutical preparations for oral use can be obtained through combining active 25 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, 3o 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.
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.
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 to 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.
Pharmaceutical formulations suitable for parentera! administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline.
Aqueous injection suspensions may contain substances which increase the viscosity of the suspension. such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil. or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
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.
The pharmaceutical compositions of the present invention may be manufactured in 3o 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.
The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder which may contain any or all of the following: I
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.
After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For i o administration of CALREP, such labeling would include amount, frequency, and method of administration.
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 t s skilled in the art.
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 3o determine useful doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient, for example CALREP or fragments thereof, antibodies of CALREP, and agonists, antagonists or inhibitors of CALREP, which ameliorates the symptoms or condition.
Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell 25 cultures or with experimental animals, such as by calculating the ED;° (the dose therapeutically effective in 50% of the population) or LD;° (the dose lethal to 50% of the population) statistics. The dose ratio of therapeutic to toxic effects is the therapeutic index, and it can be expressed as the ED;o/LD;o ratio. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays 3o 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;o 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.
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 to biweekly depending on the half life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 ,ug to 100,000 fig, 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 t 5 formulations for nucleotides than for proteins or their inhibitors.
Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
DIAGiVOSTICS
2o In another embodiment, antibodies which specif cally bind CALREP may be used for the diagnosis of disorders characterized by expression of CALREP, or in assays to monitor patients being treated with CALREP or agonists, antagonists, or inhibitors of CALREP. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for CALREP include methods 25 which utilize the antibody and a label to detect CALREP 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.
3o A variety of protocols for measuring CALREP, including ELISAs. RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of CALREP expression. Normal or standard values for CALREP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to CALREP under conditions suitable for complex formation The amount of standard complex formation may be quantitated by various methods, preferably by photometric means. Quantities of CALREP
expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding CALREP
may be used for diagnostic purposes. The polynucleotides which may be used include ~o oiigonucleotide 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 CALREP may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of CALREP, and to monitor regulation of CALREP levels during therapeutic intervention.
is In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding CALREP or closely related molecules may be used to identify nucleic acid sequences which encode CALREP.
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 2o stringency of the hybridization or amplification (maximal, high, intermediate, or low), will determine whether the probe identifies only naturally occurring sequences encoding CALREP, allelic variants, or related sequences.
Probes may also be used for the detection of related sequences, and should preferably have at least 50% sequence identity to any of the CALREP encoding sequences.
35 The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID N0:2 or from genomic sequences including promoters, enhancers, and introns of the CALREP gene.
Means for producing specific hybridization probes for DNAs encoding CALREP
include the cloning of polynucleotide sequences encoding CALREP or CALREP
3o 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 3'-P or'SS, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. and the like.
Polynucleotide sequences encoding CALREP may be used for the diagnosis of a disorder associated with expression of CALREP. Examples of such a disorder include, but are not limited to, cell proliferative disorders such as arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia; cancers including adenocarcinoma, melanoma, myeloma, sarcoma, ~ o 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 immune disorders such as leukemia, lymphoma, rheumatoid arthritis, dermatitis, inflammation, systemic lupus erythematosus and other autoimmune diseases; and neurological disorders such as akathesia, Alzheimer's disease, amnesia, amyotrophic lateral sclerosis, bipolar disorder, catatonia, cerebral neoplasms, cerebral infarctions, dementia, depression, diabetic neuropathy, Down's syndrome, tardive dyskinesia, dystonias. epilepsy, head trauma, Huntington's disease, peripheral neuropathy, multiple sclerosis, neurofibromatosis, Parkinson's disease, paranoid psychoses, postherpetic neuralgia, schizophrenia, and Tourette's disorder. The polynucleotide sequences encoding CALREP 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 CALREP expression. Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding CALREP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding CALREP may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the 3o 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 CALREP 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.
In order to provide a basis for the diagnosis of a disorder associated with expression of CALREP, 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 CALREP, under conditions suitable for hybridization or amplification. Standard to 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.
~ 5 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.
20 With respect to cancer, the presence of an abnormal 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 25 preventing the development or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences encoding CALREP 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 CALREP, or a fragment of a 3o polynucleotide complementary to the polynucleotide encoding CALREP, 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.
Methods which may also be used to quantitate the expression of CALREP include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P.C. et al.
(1993) J.
Immunol. Methods 159:235-244; and Duplaa, C. et al. (1993) Anal. Biochem. 229-236.) The speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any of s o 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.
Microarrays may be prepared. used, and analyzed using methods known in the art.
(See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M.
et al. (1996) Proc. Natl. Acad. Sci. 93:10614-10619; Baldeschweiler et al. (1995) PCT
application W095/251116; Shalom D. et al. (1995) PCT application W095/35505; Heller, R.A.
et al.
zo (1997) Proc. Natl. Acad. Sci. 94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No.
5,605.662.) In another embodiment of the invention, nucleic acid sequences encoding CALREP may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. The sequences may be mapped to a particular chromosome, ?5 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.) 3o 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 Biolo~v and Biotechnoloey, VCH
Publishers New York, NY, 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 CALREP 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.
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 t 5 a particular genomic region, e.g., ataxia-telangiectasia to 11 q22-23, 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.
20 In another embodiment of the invention, CALREP, 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 CALREP and the agent 25 being tested may be measured.
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. (I984) PCT application W084/03564.) In this method, large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some 30 other surface. The test compounds are reacted with CALREP, or fragments thereof, and washed. Bound CALREP is then detected by methods well known in the art.
Purified CALREP 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.
In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding CALREP specifically compete with a test compound for binding CALREP. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with CALREP.
In additional embodiments, the nucleotide sequences which encode CALREP 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, to including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
The examples below are provided to illustrate the subject invention and are not included for the purpose of limiting the invention.
i 5 EXAMPLES
I. cDNA Library Construction The BRAITUT03 cDNA library was constructed using RNA isolated from brain tumor tissue removed from the left frontal lobe of a 17-year-old Caucasian female during excision of a cerebral meningeal lesion. Pathology indicated a grade 4 fibrillary giant and 2o small-cell astrocytoma. Family history included benign hypertension and cerebrovascular disease.
The frozen tissue was homogenized and lysed in guanidinium isothiocyanate solution using a Brinkmann Homogenizer PoIytron-PT 3000 (Brinkmann Instruments, Westbury, NY). RNA was isolated as per Stratagene's RNA isolation protocol 25 (Stratagene, La Jolla, CA). RNA was extracted twice with acid phenol, precipitated with sodium acetate and ethanol, resuspended in RNase-free water, and treated with DNase.
Poly (A+) RNA was isolated using the Qiagen Oligotex kit (QIAGEN Inc, Chatsworth, CA).
Poly (A+) RNA was used for cDNA synthesis and construction of the 3o BRAITUT03 cDNA library according to the recommended protocols in the SuperScriptTM
plasmid system (Catalog #18248-013, GIBCO BRL, Gaithersburg, MD). cDNAs were fractionated on a Sepharose CL4B column (Catalog #275105, Pharmacia Amersham Biotech, Piscataway, NJ), and those cDNAs exceeding 400 by were ligated into pSport I
(Catalog #15382-013, G~BCO BRL). The recombinant plasmids were subsequently transformed into DHSaTM competent cells (Catalog #18258-012, G~sco BRL).
II. Isolation and Sequencing of cDNA Clones Plasmid DNA was released from the cells and purified using the REAL Prep 96 plasmid kit (Catalog #26173, QIAGEN Inc). The recommended protocol was employed except for the following changes: 1 ) the bacteria were cultured in 1 ml of sterile Terrific Broth (Catalog #22711, G~aco BRL) with carbenicillin at 25 mg/1 and glycerol at 0.4%; 2) ~o after the cultures were incubated for 19 hours, the cells were lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid DNA pellets were each resuspended in 0.1 ml of distilled water. The DNA samples were stored at 4°C.
The cDNAs were sequenced by the method of Sanger et al. ( 1975, J. Mol. Biol.
94:441 f), using a Hamilton Micro Lab 2200 (Hamilton, Reno, NV) in combination with I5 Pettier Thermal Cyclers (PTC200 from MJ Research, Watertown, MA) and Applied Biosystems 377 DNA Sequencing Systems. and the reading frame was determined.
III. Similarity Searching of cDNA Clones and Their Deduced Proteins The nucleotide sequences and/or amino acid sequences of the Sequence Listing 2o 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.. Altschui, S.F. (1993) J. Mol. Evol 36:290-300; and Altschul et al. (1990) J. Mol. Biol. 215:403-410.) 25 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 3o 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).
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-'-5 for nucleotides and 10-8 for peptides.
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.
to 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 ~ 5 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 20 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.
25 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
30 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 x 10'' 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 x 10'3 or less are considered in the functional analyses of the protein sequences in the Sequence Listing.
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.) The PFAM database contains protein sequences of 527 protein families gathered 1o 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'°°-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 ?o or comparison sequences.
Two other algorithms, SIGPEPT and TM, both based on the HMM algorithm described above (see, e.g., Eddy, s. upra; and Sonnhammer, su ra), 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 3o 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 1 I 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.
IV. Northern Analysis 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 1o 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.) Analogous computer techniques applying BLAST are used to search for identical or related molecules in nucleotide databases such as GenBank or LIFESEQTM
database (Incyte Pharmaceuticals). This analysis is much faster than multiple membrane-based t 5 hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar.
The basis of the search is the product score, which is defined as:
sequence identity x % maximum BLAST score 20 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.
25 The results of Northern analysis are reported as a list of libraries in which the transcript encoding CALREP 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.
V. Extension of CALREP Encoding Poiynucleotides The nucleic acid sequence of Incyte Clone 2109176 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 OLIGOTM 4.06 (National Biosciences, Plymouth, MN), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC
I o content of about 50% or more, and to anneal to the target sequence at temperatures of about 68°C to about 72°C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries (Gtsco BRL) were used to extend the sequence.
If more than one extension is necessary or desired, additional sets of primers are designed I s to further extend the known region.
High fidelity amplification was obtained by following the instructions for the XL-PCRTM kit (Perkin Elmer) and thoroughly mixing the enzyme and reaction mix.
PCR was performed using the Peltier Thermal Cycler (PTC200; M.J. Research. Watertown.
MA), beginning with 40 pmol of each~primer and the recommended concentrations of all other 2o components of the kit, with the following parameters:
Step 1 94 C for 1 min (initial denaturation) Step 2 65 C for 1 min Step 3 68 C for 6 min Step 4 94 C for 15 sec 2s Step 5 65 C for 1 min Step 6 68 C for 7 min Step 7 Repeat steps 4 through 6 for an additional 15 cycles Step 8 94 C for 15 sec Step 9 65 C for 1 min 3o Step 10 68 C for 7:15 min Step 11 Repeat steps 8 through 10 for an additional 12 cycles Step 12 72 C for 8 min Step 13 4 C (and holding) 35 A S ,ul to 10 ~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 _97_ were successful in extending the sequence. Bands thought to contain the largest products were excised from the gel, purified using QIAQUICKTM (QIAGEN Inc.), and trimmed of overhangs using Klenow enzyme to facilitate religation and cloning.
After ethanol precipitation, the products were redissolved in 13 ~1 of ligation buffer, l,ul T4-DNA ligase (15 units) and l,ul T4 polynucleotide kinase were added, and the mixture was incubated at room temperature for 2 to 3 hours, or overnight at 16° C.
Competent E. coli cells (in 40 ~1 of appropriate media) were transformed with 3 ~1 of ligation mixture and cultured in 80 ~1 of SOC medium. (See, e.g., Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37°C, the E. coli mixture was plated to on Luria Bertani (LB) agar (See, e.g., Sambrook, supra, Appendix A, p. 1) containing carbenicillin (2x carb). The following day, several colonies were randomly picked from each plate and cultured in 150 ul of liquid LB/2x Garb medium placed in an individual well of an appropriate commercially-available sterile 96-well microtiter plate. The following day, ~ ul of each overnight culture was transferred into a non-sterile 96-well plate and, after dilution 1:10 with water, 5 ul from each sample was transferred into a PCR
array.
For PCR amplification. 18 ,ul of concentrated PCR reaction mix (3.3x) 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 2o performed using the following conditions:
Step 1 94 C for 60 sec Step 2 94 C for 20 sec Step 3 SS C for 30 sec Step 4 72 C for 90 sec Step S Repeat steps 2 through 4 for an additional 29 cycles Step 6 72 C for 180 sec Step 7 4 C (and holding) Aliquots of the PCR reactions were run on agarose gels together with molecular 3o 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.
In like manner, the nucleotide sequence of SEQ ID N0:2 is used to obtain 5' regulatory sequences using the procedure above. oligonucleotides designed for 5' extension, and an appropriate genomic library.
_48_ VI. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID N0:2 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 OLIGOT'" 4.06 software (National Biosciences) and labeled by combining SO
pmol of each oligomer, 250 ~Ci of [y-''-P] adenosine triphosphate (Amersham, Chicago, IL), and T4 polynucleotide kinase (DuPont NEN~, Boston, MA). The labeled oligonucleotides are substantially purified using a SephadexTM G-25 superfine size exclusion dextran bead column (Pharmacia & Upjohn. Kalamazoo, MI). An aliquot containing 10' counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xbal, or Pvu II (DuPont NEN, Boston, MA).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham, NH).
Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT ARTM film (Kodak, Rochester, NY) is exposed to the blots to film for several hours, hybridization patterns are 3o compared visually.
VII. Microarrays 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 35 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 30 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.
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 LASERGENETM. 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 Shalom 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.
VIII. Complementary Polynucleotides Sequences complementary to the CALREP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring CALREP.
~5 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 OLIGOT"' 4.06 software and the coding sequence of CALREP. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the 2o coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the CALREP-encoding transcript.
IX. Expression of CALREP
Expression and purification of CALREP is achieved using bacterial or virus-based 25 expression systems. For expression of CALREP 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 TS or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are 30 transformed into suitable bacterial hosts, e.g., BL21(DE3). Antibiotic resistant bacteria express CALREP upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG).
Expression of CALREP 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 CALREP 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 (Sf~) 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.
to Gene Ther. 7:1937-1945.) In most expression systems, CALREP 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, t5 enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Pharmacia, Piscataway, NJ).
Following purification, the GST moiety can be proteolytically cleaved from CALREP at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman 2o Kodak. Rochester, NY). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN Inc, Chatsworth, CA). Methods for protein expression and purification are discussed in Ausubeh F. M. et al. ( 1995 and periodic supplements) Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York, NY, ch 10, 16. Purified CALREP obtained by these methods can be used directly in the 25 following activity assay.
X. Demonstration of CALREP Activity An assay for CALREP activity measures the binding of CALREP to Ca'-' using a Ca'-' overlay system. (Weis, K. et al. (1994) J. Biol. Chem. 269:19142-19150.) Purified 3o CALREP is transferred and immobilized onto a nitrocellulose membrane. The membrane is washed three times with buffer (60 mM KC1, 5 mM MgCI,, 10 mM imidazole-HCI, pH
6.8) and incubated in this buffer for 10 minutes with 1 pCi [~SCa'-~] (NEN-DuPont, Boston, -sl-MA). Unbound [45Ca'-+] is removed from the membrane by washing with water, and the membrane is dried. Membrane-bound [~SCa'--J is detected by autoradiography and quantified using image analysis systems and software. CALREP activity is proportional to the amount of [~SCa'-~] detected on the membrane.
XI. Functional Assays CALREP function is also assessed by expressing the sequences encoding CALREP at physiologically elevated levels in mammalian cell culture systems.
cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives to high levels of cDNA expression. Vectors of choice include pCMV SPORTTM
(Life Technologies, Gaithersburg, MD) and pCRTM 3.1 (Invitrogen, Carlsbad, CA, both of which contain the cytomegalovirus promoter. 5-10 ~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 ~,g of an additional t 5 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, CA), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated.
laser 20 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 25 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 3o discussed in Ormerod, M. G. (1994) Flow Cytometry, Oxford, New York, NY.
The influence of CALREP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding CALREP 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, NY). mRNA can be purified from the cells using methods well known by those of skill in the art.
Expression of mRNA encoding CALREP and other genes of interest can be analyzed by Northern analysis or microarray techniques.
XII. Production of CALREP Specific Antibodies 1o CALREP substantially purified using polyacrylamide gel electrophoresis (PAGE)(see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the CALREP amino acid sequence is analyzed using ~ 5 LASERGENETM software (DNASTAR Inc.) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel supra, ch. 11.) 2o Typically, oligopeptides 1 S residues in length are synthesized using an Applied Biosystems Peptide Synthesizer Model 431 A 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 su ra.) Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant.
Resulting 25 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.
XIII. Purification of Naturally Occurring CALREP Using Specific Antibodies 3o Naturally occurring or recombinant CALREP is substantially purified by immunoaffinity chromatography using antibodies specific for CALREP. An imrnunoaffinity column is constructed by covalently coupling anti-CALREP
antibody to an activated chromatographic resin, such as CNBr-activated Sepharose (Pharmacia &
Upjohn). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing CALREP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of CALREP
(e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/CALREP 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 CALREP is collected.
1o XIV. Identification of Molecules Which Interact with CALREP
CALREP, or biologically active fragments thereof, are labeled with''-SI
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 CALREP, washed, and any wells with labeled CALREP complex are assayed.
Data obtained using different concentrations of CALREP are used to calculate values for the number, affinity. and association of CALREP with the candidate molecules.
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 2o 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 LISTING
<110> INCYTE PHARMACEUTICALS, INC.
TANG, Y. Tom CORLEY, Neil C.
GUEGLER, Karl J.
PATTERSON, Chandra <120> CALMODULIN RELATED PROTEIN
<130> PF-0528 PCT
<140> To Be Assigned <141> Herewith <150> 09/083,908 <151> 1998-05-22 <160> 3 <170> Perl Program <210> 1 <2I1> 386 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte clone 2109176 <400> 1 Met Tyr Gly Glu Cys Arg Thr Tyr Ile Ile His Tyr Tyr Leu Met Asp Asp Thr Val Glu Ile Arg Glu Val His Glu Arg Asn Asp Gly Arg Asp Pro Phe Pro Leu Leu Met Asn Arg Gln Arg Val Pro Lys Val Leu Val Glu Asn Ala Lys Asn Phe Pro Gln Cys Val Leu Glu Ile Ser Asp Gln Glu Val Leu Glu Trp Tyr Thr Ala Lys Asp Phe Ile Val Gly Lys Ser Leu Thr Ile Leu Gly Arg Thr Phe Phe Ile Tyr Asp Cys Asp Pro Phe Thr Arg Arg Tyr Tyr Lys Glu Lys Phe Gly Ile Thr Asp Leu Pro Arg Ile Asp Val Ser Lys Arg Glu Pro Pro Pro Val Lys Gln Glu Leu Pro Pro Tyr Asn Gly Phe Gly Leu Val Glu Asp Ser Ala Gln Asn Cys Phe Ala Leu Ile Pro Lys Ala Pro Lys Lys Asp Val Ile Lys Met Leu Val Asn Asp Asn Lys Val Leu Arg Tyr Leu Ala Val Leu Glu Ser Pro Ile Pro Glu Asp Lys Asp Arg Arg Phe Val Phe Ser Tyr Phe Leu Ala Thr Asp Met Ile Ser Ile Phe Glu Pro Pro Val Arg Asn Ser Gly Ile Ile Gly Gly Lys Tyr Leu Gly Arg Thr Lys Val Val Lys Pro Tyr Ser Thr Val Asp Asn Pro Val Tyr Tyr Gly Pro Ser Asp Phe Phe Ile Gly Ala Val Ile Glu Val Phe Gly His Arg Phe Ile Ile Leu Asp Thr Asp Glu Tyr Val Leu Lys Tyr Met Glu Ser Asn Ala Ala Gln Tyr Ser Pro Glu Ala Leu Ala Ser Ile Gln Asn His Val Arg Lys Arg Glu Ala Pro Ala Pro Glu Ala Glu Ser Lys Gln Thr Glu Lys Asp Pro Gly Val Gln Glu Leu Glu Ala Leu Ile Asp Thr Ile Gln Lys Gln Leu Lys Asp His Ser Cys Lys Asp Asn Ile Arg Glu Ala Phe Gln Ile Tyr Asp Lys Glu Ala Ser Gly Tyr Val Asp Arg Asp Met Phe Phe Lys Ile Cys Glu Ser Leu Asn Val Pro Val Asp Asp Ser Leu Val Lys Glu Leu Ile Arg Met Cys Ser His Gly Glu Gly Lys Ile Asn Tyr Tyr Asn Phe Val Arg Ala Phe Ser Asn <210> 2 <211> 1338 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte clone 2109176 <400> 2 cccatcagac tttgatcaac tcaagcaatt tctcaccttt gacaaacagg tccttcgatt 60 ctatgcaatc tgggatgata cagacagcat gtatggtgaa tgtcggacct acatcattca 120 ttactatctt atggatgata cggtggaaat tcgagaggtc cacgaacgga atgatgggag 180 agatcctttc ccactcctaa tgaaccgcca gcgtgtgccc aaagttttgg tggaaaatgc 240 aaagaacttc cctcagtgtg tgctagaaat ctctgaccaa gaagtgttgg aatggtatac 300 tgctaaagac ttcattgttg ggaagtcact cactatcctt gggagaactt tcttcattta 360 tgattgtgat ccatttactc gacggtatta caaagagaag tttggaatca ctgatttacc 420 acgtattgat gtgagcaagc gggaaccacc tccagtaaaa caggagttgc ctccttataa 480 cggttttgga ctagtggaag attctgctca gaattgtttt gctctcattc caaaagctcc 540 aaaaaaagac gttattaaaa tgctggtgaa tgataacaag gtgcttcgtt atttggctgt 600 actggaatcc cccatcccag aagacaaaga ccgcagattt gtcttctctt actttctagc 660 taccgacatg atcagtatct ttgagcctcc tgttcgcaat tctggtatca ttgggggcaa 720 gtaccttggc aggactaaag ttgttaaacc atactctaca gtggacaacc ctgtctacta 780 tggccccagt gacttcttca ttggtgctgt gattgaagtg tttggtcacc ggttcatcat 840 ccttgataca gacgagtatg ttttgaaata catggagagc aacgctgccc agtattcacc 900 2l3 agaagcactc gcgtcaattc agaaccatgt ccgaaagcga gaagcgcctg ctccagaagc 960 agaaagcaag caaactgaaa aggatccagg cgtgcaggaa ttggaagcat taatagacac 1020 aattcagaag caactgaaag atcactcatg caaagacaac attcgtgagg catttcaaat 1080 ttatgacaag gaagcttcag gatatgtgga cagagacatg ttctttaaaa tctgtgaatc 1140 gcttaacgtc ccagtggatg actccttggt taaggagtta atcaggatgt gctctcatgg 1200 agaaggcaaa attaactact ataactttgt tcgtgctttc tcaaactgac ctgctgatga 1260 gaaaatgcaa gacaattttt gatactggaa ctatgctttg aaatacacct tacactcttc 1320 atagaggcaa aaaaaaaa 1338 <210> 3 <211> 149 <212> PRT
<213> Homo sapiens <300>
<308> 8189081 <400> 3 Met Ala Asp Gln Leu Thr Glu Glu Gln Val Thr Glu Phe Lys Glu Ala Phe Ser Leu Phe Asp Lys Asp Gly Asp Gly Cys Ile Thr Thr Arg Glu Leu Gly Thr Val Met Arg Ser Leu Gly Gln Asn Pro Thr Glu Ala Glu Leu Arg Asp Met Met Ser Glu Ile Asp Arg Asp Gly Asn Gly Thr Val Asp Phe Pro Glu Phe Leu Gly Met Met Ala Arg Lys Met Lys Asp Thr Asp Asn Glu Glu Glu Ile Arg Glu Ala Phe Arg Val Phe Asp Lys Asp Gly Asn Gly Phe Val Ser Ala Ala Glu Leu Arg His Val Met Thr Arg Leu Gly Glu Lys Leu Ser Asp Glu Glu Val Asp Glu Met Ile Arg Ala Ala Asp Thr Asp Gly Asp Gly Gln Val Asn Tyr Glu Glu Phe Val Arg Val Leu Val Ser Lys
The term "agonist," as used herein, refers to a molecule which, when bound to CALREP, increases or prolongs the duration of the effect of CALREP. Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of CALREP.
to An "allelic variant," as this term is used herein, is an alternative form of the gene encoding CALREP. 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 ~ 5 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.
"Altered" nucleic acid sequences encoding CALREP, as described herein, include those sequences with deletions, insertions, or substitutions of different nucleotides, 2o resulting in a polynucleotide the same as CALREP or a polypeptide with at least one functional characteristic of CALREP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding CALREP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide 25 sequence encoding CALREP. 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 CALREP. 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 3o the biological or immunological activity of CALREP 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.
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 CALREP which are preferably about 5 to about I S amino acids in length, most preferably 14 amino acids, and which retain some biological activity or immunological activity of CALREP. Where "amino t o 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.
"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, NY, pp.l-5.) The term "antagonist," as it is used herein, refers to a molecule which, when bound 2o to CALREP, decreases the amount or the duration of the effect of the biological or immunological activity of CALREP. Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules which decrease the effect of CALREP.
As used herein, the term "antibody" refers to intact molecules as well as to fragments thereof, such as Fab, F(ab'),, and Fv fragments, which are capable of binding the epitopic determinant. Antibodies that bind CALREP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers 3o that are chemically coupled to peptides include bovine serum albumin, thyroglobuiin, 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.
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.
i 5 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 CALREP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
2o The terms "complementary" or ''complementarity," as used herein, refer to the natural binding of polynucleotides 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 25 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.
3o 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 _8_ composition may comprise a dry formulation, an aqueous solution, or a sterile composition. Compositions comprising polynucleotide sequences encoding CALREP
or fragments of CALREP 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 s carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts, e.g., NaCI, detergents, e.g.,sodium dodecyl sulfate (SDS), and other components, e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
"Consensus sequence," as used herein, refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, extended using XL-PCRTM (Perkin Elmer, Norwalk, CT) 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 GELVIEWTM Fragment Assembly system (GCG, Madison, WI). Some sequences have been both extended and assembled to produce the consensus sequence.
~ 5 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 CALREP, by Northern analysis is indicative of the presence of nucleic acids encoding CALREP in a sample, and thereby correlates with expression of the transcript from the polynucleotide encoding CALREP.
20 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.
The term "derivative," as used herein, refers to the chemical modification of a polypeptide sequence, or a polynucleotide sequence. Chemical modifications of a 25 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 3o was derived.
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 t o 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.
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 MegAlignTM program (DNASTAR, Inc., Madison WI). The MegAlignT"' 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.
183:62b-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions.
"Human artificial chromosomes" (HACs), as described herein, are linear -lo-microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain ali 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.) 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.
"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.
to 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., Cot or Rot 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).
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.
"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.
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.
The terms "element" or "array element" as used herein in a microarray context, refer to hybridizable polynucleotides arranged on the surface of a substrate.
The term "modulate," as it appears herein, refers to a change in the activity of CALREP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of CALREP.
-il-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.
The terms "operably associated" or "operably linked," as used herein, refer to 1o 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 t5 operator sequences that control expression of the polypeptide.
The term "oligonucleotide," as used herein, refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 1 ~ 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 ?o substantially equivalent to the terms "amplimer," "primer." "oligomer," and "probe." as these terms are commonly defined in the art.
"Peptide nucleic acid" (PNA), as used herein, refers to an antisense molecule or anti-gene agent which comprises an oiigonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine.
The terming!
25 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.) The term "sample," as used herein, is used in its broadest sense. A biological 3o sample suspected of containing nucleic acids encoding CALREP, or fragments thereof, or CALREP 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.
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.
As used herein, the term "stringent conditions" refers to conditions which permit to 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.
~ 5 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.
A "substitution," as used herein, refers to the replacement of one or more amino 2o acids or nucleotides by different amino acids or nucleotides, respectively.
"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 25 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 3o chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
A ''variant" of CALREP. 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, LASERGENETM software.
THE INVENTION
The invention is based on the discovery of a new human calmodulin related protein (CALREP), the polynucleotides encoding CALREP, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative and neurological disorders.
Nucleic acids encoding the CALREP of the present invention were first identified in Incyte Clone 2109176 from the brain tumor cDNA library (BRAITUT03) using a computer search, e.g., BLAST, for amino acid sequence alignments. A consensus sequence, SEQ ID N0:2, was derived from the following overlapping and/or extended nucleic acid sequences: Incyte Clones 2109176 (BRAITUT03), 2653689 (THYMNOT04), 2878785 (UTRSTUTOS), and 959509 (BRSTTUT03).
2o In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:1, as shown in Figures 1 A. 1 B, 1 C, and 1 D.
CALREP is 386 amino acids in length and has eight potential casein kinase II
phosphorylation sites at 562, T71, S116, S196, S223. T296, S320, and 5369, and five potential protein kinase C phosphorylation sites at T71, T97, 5116, T296, and 5320. In 35 addition, both BLOCKS and PFAM analyses reveal an EF-hand motif within the region from about N324 to L352. Positions 1, 3, S, 7, and 9 of the EF-hand consensus motif are conserved in CALREP at D333, E335, S337, Y339, and D341. The conserved glycine at position 6 of the EF-hand consensus motif is also present in CALREP at 6338.
Secondary structure analysis also indicates that the regions flanking the putative EF-hand are 3o predicted to form a-helices. As shown in Figure 2, the EF-hand region of CALREP has chemical and structural similarity with the region comprising the third EF-hand of NB-1 (GI 189081; SEQ ID N0:3). Note that in Figure 2, only the relevant EF-hand regions of WO 99/61b09 PCT/US99/10747 CALREP and NB-1 are shown, and the amino acid residues are numbered based on their positions in the complete protein sequences as reported in the Sequence Listing. CALREP
and NB-1 share 30% identity within the regions shown. In addition, the potential phosphorylation sites at S196 and S369 of CALREP are conserved in NB-1. A
region of unique sequence in CALREP from about amino acid 21 to about amino acid 30 is encoded by a fragment of SEQ ID N0:2 from about nucleotide 149 to about nucleotide 178.
Northern analysis shows the expression of this sequence in various libraries, at least 67%
of which are associated with cancerous or proliferating tissue and at least 39% of which are associated with the immune response. In addition, 22% of the libraries expressing t o CALREP are derived from reproductive tissue, and 22% are derived from neurological tissue, particularly Alzheimer's and tumor-associated brain tissue.
The invention also encompasses CALREP variants. A preferred CALR.EP 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 CALREP amino acid ~ 5 sequence, and which contains at least one functional or structural characteristic of CALREP.
The invention also encompasses polynucleotides which encode CALREP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising the sequence of SEQ ID N0:2, which encodes an CALREP.
2o The invention also encompasses a variant of a polynucleotide sequence encoding CALREP. 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 CALREP. A
particular aspect of the invention encompasses a variant of SEQ ID N0:2 which has at 25 least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to SEQ ID N0:2. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of CALREP.
It will be appreciated by those skilled in the art that as a result of the degeneracy of 3o the genetic code. a multitude of polynucieotide sequences encoding CALREP, 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 CALREP, and all such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode CALREP and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring CALREP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding CALREP or its derivatives possessing a ~o 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 CALREP and its derivatives without altering the encoded t 5 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.
The invention also encompasses production of DNA sequences which encode CALREP and CALREP derivatives, or fragments thereof, entirely by synthetic chemistry.
3o 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 CALREP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are capable 25 of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID N0:2, or a fragment of SEQ ID N0:2, under various conditions of stringency.
(See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407;
Kimmel, A.R. (1987) Methods Enzymol. 152:507-511.) For example, stringent salt concentration will ordinarily be less than about 750 mM NaCI and 75 mM trisodium citrate, preferably 30 less than about 500 mM NaCI and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCI 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°C, more preferably of at least about 37°C, and most preferably of at least about 42°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°C in 750 mM NaCI, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37°C in S00 mM NaCI, 50 mM trisodium citrate, 1% SDS, 35%
formamide, and 100 ~cg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42°C in 250 mM NaCI, 25 mM trisodium citrate, 1% SDS, 50 formamide, and 200 ~cg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
~5 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 NaCI and 3 mM trisodium citrate, and most preferably less than zo about 15 mM NaCI and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include temperature of at least about 25°C, more preferably of at least about 42°C, and most preferably of at least about 68°C. In a preferred embodiment, wash steps will occur at 25°C in 30 mM NaCI, 3 mM trisodium citrate. and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42°C
in 15 mM
z5 NaCI, 1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C in 15 mM NaCI, 1.5 mM trisodium citrate, and 0.1% SDS.
Additional variations on these conditions will be readily apparent to those skilled in the art.
Methods for DNA sequencing are well known and generally available in the art 3o 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~ (US
Biochemical Corp., Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable -m-polymerase (Amersham,. Chicago, IL), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASETM Amplification System (Gteco BRL, Gaithersburg, MD). Preferably, the process is automated with machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), Peltier Thermal Cycler (PTC200;
MJ
Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
The nucleic acid sequences encoding CALREP 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 t o 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 t5 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 at. (1991) PCR Methods Applic. 1:111-119.) In this method. multiple restriction enzyme digestions and ligations may be used to insert an 2o 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 PromoterFinderTM libraries to walk genomic DNA (Clontech, Palo Alto, CA). This procedure avoids the need to screen 25 libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGOTM
4.06 Primer Analysis software (National Biosciences Inc., Plymouth, MN) 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°C to 72°C.
3o 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.
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., GenotyperT"' and Sequence NavigatorTM, Perkin Elmer), and the entire process from ~ o 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.
In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode CALREP may be cloned in recombinant DNA molecules that direct ~ 5 expression of CALREP, 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 CALREP.
The nucleotide sequences of the present invention can be engineered using 2o methods generally known in the art in order to alter CALREP-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 25 may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
In another embodiment, sequences encoding CALREP may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al. ( 1980) Nucl. Acids Res. Symp. Ser. 215-223, and Horn, T. et al. ( 1980) Nucl.
3o Acids Res. Symp. Ser. 225-232.) Alternatively, CALREP 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 431 A Peptide Synthesizer (Perkin Elmer). Additionally, the amino acid sequence of CALREP, 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.
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, NY.) In order to express a biologically active CALREP, the nucleotide sequences encoding CALREP 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 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding CALREP. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding CALREP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding CALREP and its initiation codon 20 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 25 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.) Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding CALREP and appropriate 3o 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, NY, ch. 4, 8, and 16-17; and Ausubel. F.M. et al. (1995, and periodic supplements) Current Protocols in Molecular BioloQV, John Wiley &
Sons, New York, NY, ch. 9, 13, and 16.) A variety of expression vector/host systems may be utilized to contain and express sequences encoding CALREP. 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 t0 vines (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.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding CALREP.
For example, routine cloning, subcloning, and propagation of polynucleotide sequences ~ 5 encoding CALREP can be achieved using a multifunctional E. coli vector such as Bluescript~ (Stratagene) or pSportlTM plasmid (Gt6co BRL). Legation of sequences encoding CALREP into the vector's multiple cloning site disrupts the lacZ
gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition. these vectors may be useful for in vitro transcription, 2o 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 CALREP are needed, e.g.
for the production of antibodies, vectors which direct high level expression of CALREP
may be used. For example, vectors containing the strong, inducible T5 or T7 bacteriophage 25 promoter may be used.
Yeast expression systems may be used for production of CALREP. 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~astoris.
In addition, such vectors direct either the secretion or intracellular retention of expressed 3o 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.) Plant systems may also be used for expression of CALREP. Transcription of sequences encoding CALREP 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 aI. (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 to Yearbook of Science and Technolo~y (1992) McGraw Hill, New York, NY; 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 CALREP may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E 1 or E3 region of the viral genome may be used to obtain infective virus which expresses CALREP 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.
2o 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.
For long term production of recombinant proteins in mammalian systems, stable expression of CALREP in cell lines is preferred. For example, sequences encoding CALREP 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 3o 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.
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- or apr 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 G-418; and als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F. et al {1981) J. Mol.
Biol. 150:1-14; and Murry, s_unra.) 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, CA),13 glucuronidase and its substrate 13-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.
X5:121-131.) Although the presence/absence of marker gene expression suggests that the gene of ?o interest is also present, the presence and expression of the gene may need to be confirmed.
For example, if the sequence encoding CALREP is inserted within a marker gene sequence, transformed cells containing sequences encoding CALREP can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding CALREP 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.
In general, host cells that contain the nucleic acid sequence encoding CALREP
and that express CALREP 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
3o 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.
WO 99/61609 PCTNS99/10?47 Immunological methods for detecting and measuring the expression of CALREP
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 CALREP 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, MN, Section IV;
Coligan, J. E. et al. ( 1997 and periodic supplements) Current Protocols in Immunolo~y, to Greene Pub. Associates and Wiley-Interscience, New York, NY; and Maddox, D.E. et al.
(1983) J. Exp. Med. 158:1211-1216).
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 CALREP include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding CALREP, 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 2o such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Pharmacia & Upjohn (Kalamazoo, MI), Promega (Madison, WI), and U.S. Biochemical Corp. (Cleveland, OH).
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.
Host cells transformed with nucleotide sequences encoding CALREP 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 CALREP may be designed to contain signal sequences which direct secretion of CALREP
through a prokaryotic or eukaryotic cell membrane.
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, HEK293, and WI38), are available from the American Type Culture Collection (ATCC, Bethesda, MD) and may be chosen to ensure the correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding CALREP 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 CALREP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of CALREP 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, 2o 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 CALREP encoding sequence and the heterologous protein sequence, so that CALREP 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 3o Molecular Biolo~y, John Wiley & Sons, New York, NY, ch 10. A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
In a further embodiment of the invention. synthesis of radiolabeled CALREP may be achieved in vitro using the TNTTM rabbit reticulocyte lysate or wheat germ extract systems (Promega, Madison, WI). 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 3sS-methionine.
Fragments of CALREP 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 Peptide Synthesizer (Perkin Elmer). Various fragments of CALREP may be synthesized separately and then combined to produce the full length molecule.
THERAPEUTICS
Chemical and structural similarity exists between CALREP and the EF-hand domain, particularly that of NB-1 from human (GI 189081). In addition, CALREP
is expressed in proliferating and neurological tissue. Therefore, CALREP appears to play a role in cell proliferative and neurological disorders.
Therefore, in one embodiment, CALREP or a fragment or derivative thereof may ?o be administered to a subject to treat or prevent a cell proliferative disorder associated with decreased expression of CALREP. Such disorders can include, but are not limited to, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia; cancers including adenocarcinoma, 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 immune disorders such as leukemia, lymphoma, rheumatoid arthritis, dermatitis, inflammation, systemic lupus erythematosus and other autoimmune diseases.
In another embodiment, a vector capable of expressing CALREP or a fragment or derivative thereof may be administered to a subject to treat or prevent a cell proliferative disorder including, but not limited to, those described above.
In a further embodiment, a pharmaceutical composition comprising a substantially purified CALREP in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a cell proliferative disorder including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of CALREP
may be administered to a subject to treat or prevent a cell proliferative disorder including, but not limited to, those listed above.
In a further embodiment, an antagonist of CALREP may be administered to a subject to treat or prevent a cell proliferative disorder associated with increased expression of CALREP. Such disorders can include, but are not limited to, those discussed above. In one aspect. an antibody which specifically binds CALREP 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 CALREP.
t 5 In an additional embodiment, a vector expressing the complement of the polynucleotide encoding CALREP may be administered to a subject to treat or prevent a cell proliferative disorder including, but not limited to, those described above.
In another embodiment, CALREP or a fragment or derivative thereof may be administered to a subject to treat or prevent a neurological disorder associated with 30 decreased expression of CALREP. Such disorders can include. but are not limited to, akathesia, Alzheimer's disease, amnesia, amyotrophic lateral sclerosis, bipolar disorder, catatonia, cerebral neoplasms, cerebral infarctions, dementia, depression, diabetic neuropathy, Down's syndrome, tardive dyskinesia, dystonias, epilepsy, head trauma, Huntington's disease, peripheral neuropathy, multiple sclerosis, neurofibromatosis, 25 Parkinson's disease, paranoid psychoses, postherpetic neuralgia, schizophrenia, and Tourette's disorder.
In another embodiment, a vector capable of expressing CALREP or a fragment or derivative thereof may be administered to a subject to treat or prevent a neurological disorder including, but not limited to, those described above.
3o In a further embodiment, a pharmaceutical composition comprising a substantially purified CALREP in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a neurological disorder including, but not _2~_ limited to. those provided above.
In still another embodiment, an agonist which modulates the activity of CALREP
may be administered to a subject to treat or prevent a neurological disorder including, but not limited to, those listed above.
In a further embodiment, an antagonist of CALREP may be administered to a subject to treat or prevent a neurological disorder associated with increased expression of CALREP. Such disorders can include, but are not limited to, those discussed above. In one aspect, an antibody which specifically binds CALREP may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical I o agent to cells or tissue which express CALREP.
In an additional embodiment. a vector expressing the complement of the polynucleotide encoding CALREP may be administered to a subject to treat or prevent a neurological disorder including, but not limited to, those described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists, I S 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 2o 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.
An antagonist of CALREP may be produced using methods which are generally known in the art. In particular, purified CALREP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind 25 CALREP. Antibodies to CALREP 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.
30 For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans. and others may be immunized by injection with CALREP 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.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to CALREP 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 to 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 CALREP amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
Monoclonal antibodies to CALREP 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.) 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 CALREP-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.) 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.) Antibody fragments which contain specific binding sites for CALREP 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 !o 246:1275-1281.) 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 !5 formation between CALREP and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering CALREP
epitopes is preferred, but a competitive binding assay may also be employed.
(Maddox, supra. ) In another embodiment of the invention, the polynucleotides encoding CALREP, 20 or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect. the complement of the polynucleotide encoding CALREP 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 CALREP. Thus, complementary molecules or fragments may be used to 25 modulate CALREP 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 CALREP.
Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia 3o 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 CALREP. (See, e.g., Sambrook, suyra; and Ausubel, s, unra.) Genes encoding CALREP can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide, or fragment thereof, encoding CALREP. 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 i o system.
As mentioned above, modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA. RNA, or PNA) to the control, S', or regulatory regions of the gene encoding CALREP.
Oligonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +10 from the t5 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, ?o B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt.
Kisco. NY, pp. I63-177.) A complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific 25 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 endonucieolytic cleavage of sequences encoding CALREP.
3o 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 WO 99/61b09 PCT/US99/10747 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.
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 chemic2l synthesis. Alternatively, RNA molecules may be generated by ! o in vitro and in vivo transcription of DNA sequences encoding CALREP. 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.
!5 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 2o 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.
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 25 introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al. ( 1997) Nature Biotechnology 15:462-466.) Any of the therapeutic methods described above may be applied to any subject in 3o need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
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 CALREP, antibodies to CALREP, and mimetics, agonists, antagonists, or inhibitors of CALREP. 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.
The pharmaceutical compositions utilized in this invention may be administered by t o 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.
In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries ~ 5 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 Remin~ton's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).
Pharmaceutical compositions for oral administration can be formulated using 2o 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.
Pharmaceutical preparations for oral use can be obtained through combining active 25 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, 3o 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.
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.
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 to 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.
Pharmaceutical formulations suitable for parentera! administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline.
Aqueous injection suspensions may contain substances which increase the viscosity of the suspension. such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil. or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
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.
The pharmaceutical compositions of the present invention may be manufactured in 3o 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.
The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder which may contain any or all of the following: I
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.
After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For i o administration of CALREP, such labeling would include amount, frequency, and method of administration.
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 t s skilled in the art.
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 3o determine useful doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient, for example CALREP or fragments thereof, antibodies of CALREP, and agonists, antagonists or inhibitors of CALREP, which ameliorates the symptoms or condition.
Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell 25 cultures or with experimental animals, such as by calculating the ED;° (the dose therapeutically effective in 50% of the population) or LD;° (the dose lethal to 50% of the population) statistics. The dose ratio of therapeutic to toxic effects is the therapeutic index, and it can be expressed as the ED;o/LD;o ratio. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays 3o 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;o 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.
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 to biweekly depending on the half life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 ,ug to 100,000 fig, 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 t 5 formulations for nucleotides than for proteins or their inhibitors.
Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
DIAGiVOSTICS
2o In another embodiment, antibodies which specif cally bind CALREP may be used for the diagnosis of disorders characterized by expression of CALREP, or in assays to monitor patients being treated with CALREP or agonists, antagonists, or inhibitors of CALREP. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for CALREP include methods 25 which utilize the antibody and a label to detect CALREP 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.
3o A variety of protocols for measuring CALREP, including ELISAs. RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of CALREP expression. Normal or standard values for CALREP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to CALREP under conditions suitable for complex formation The amount of standard complex formation may be quantitated by various methods, preferably by photometric means. Quantities of CALREP
expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding CALREP
may be used for diagnostic purposes. The polynucleotides which may be used include ~o oiigonucleotide 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 CALREP may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of CALREP, and to monitor regulation of CALREP levels during therapeutic intervention.
is In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding CALREP or closely related molecules may be used to identify nucleic acid sequences which encode CALREP.
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 2o stringency of the hybridization or amplification (maximal, high, intermediate, or low), will determine whether the probe identifies only naturally occurring sequences encoding CALREP, allelic variants, or related sequences.
Probes may also be used for the detection of related sequences, and should preferably have at least 50% sequence identity to any of the CALREP encoding sequences.
35 The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID N0:2 or from genomic sequences including promoters, enhancers, and introns of the CALREP gene.
Means for producing specific hybridization probes for DNAs encoding CALREP
include the cloning of polynucleotide sequences encoding CALREP or CALREP
3o 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 3'-P or'SS, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. and the like.
Polynucleotide sequences encoding CALREP may be used for the diagnosis of a disorder associated with expression of CALREP. Examples of such a disorder include, but are not limited to, cell proliferative disorders such as arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia; cancers including adenocarcinoma, melanoma, myeloma, sarcoma, ~ o 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 immune disorders such as leukemia, lymphoma, rheumatoid arthritis, dermatitis, inflammation, systemic lupus erythematosus and other autoimmune diseases; and neurological disorders such as akathesia, Alzheimer's disease, amnesia, amyotrophic lateral sclerosis, bipolar disorder, catatonia, cerebral neoplasms, cerebral infarctions, dementia, depression, diabetic neuropathy, Down's syndrome, tardive dyskinesia, dystonias. epilepsy, head trauma, Huntington's disease, peripheral neuropathy, multiple sclerosis, neurofibromatosis, Parkinson's disease, paranoid psychoses, postherpetic neuralgia, schizophrenia, and Tourette's disorder. The polynucleotide sequences encoding CALREP 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 CALREP expression. Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding CALREP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding CALREP may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the 3o 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 CALREP 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.
In order to provide a basis for the diagnosis of a disorder associated with expression of CALREP, 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 CALREP, under conditions suitable for hybridization or amplification. Standard to 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.
~ 5 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.
20 With respect to cancer, the presence of an abnormal 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 25 preventing the development or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences encoding CALREP 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 CALREP, or a fragment of a 3o polynucleotide complementary to the polynucleotide encoding CALREP, 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.
Methods which may also be used to quantitate the expression of CALREP include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P.C. et al.
(1993) J.
Immunol. Methods 159:235-244; and Duplaa, C. et al. (1993) Anal. Biochem. 229-236.) The speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any of s o 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.
Microarrays may be prepared. used, and analyzed using methods known in the art.
(See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M.
et al. (1996) Proc. Natl. Acad. Sci. 93:10614-10619; Baldeschweiler et al. (1995) PCT
application W095/251116; Shalom D. et al. (1995) PCT application W095/35505; Heller, R.A.
et al.
zo (1997) Proc. Natl. Acad. Sci. 94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No.
5,605.662.) In another embodiment of the invention, nucleic acid sequences encoding CALREP may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. The sequences may be mapped to a particular chromosome, ?5 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.) 3o 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 Biolo~v and Biotechnoloey, VCH
Publishers New York, NY, 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 CALREP 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.
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 t 5 a particular genomic region, e.g., ataxia-telangiectasia to 11 q22-23, 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.
20 In another embodiment of the invention, CALREP, 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 CALREP and the agent 25 being tested may be measured.
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. (I984) PCT application W084/03564.) In this method, large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some 30 other surface. The test compounds are reacted with CALREP, or fragments thereof, and washed. Bound CALREP is then detected by methods well known in the art.
Purified CALREP 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.
In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding CALREP specifically compete with a test compound for binding CALREP. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with CALREP.
In additional embodiments, the nucleotide sequences which encode CALREP 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, to including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
The examples below are provided to illustrate the subject invention and are not included for the purpose of limiting the invention.
i 5 EXAMPLES
I. cDNA Library Construction The BRAITUT03 cDNA library was constructed using RNA isolated from brain tumor tissue removed from the left frontal lobe of a 17-year-old Caucasian female during excision of a cerebral meningeal lesion. Pathology indicated a grade 4 fibrillary giant and 2o small-cell astrocytoma. Family history included benign hypertension and cerebrovascular disease.
The frozen tissue was homogenized and lysed in guanidinium isothiocyanate solution using a Brinkmann Homogenizer PoIytron-PT 3000 (Brinkmann Instruments, Westbury, NY). RNA was isolated as per Stratagene's RNA isolation protocol 25 (Stratagene, La Jolla, CA). RNA was extracted twice with acid phenol, precipitated with sodium acetate and ethanol, resuspended in RNase-free water, and treated with DNase.
Poly (A+) RNA was isolated using the Qiagen Oligotex kit (QIAGEN Inc, Chatsworth, CA).
Poly (A+) RNA was used for cDNA synthesis and construction of the 3o BRAITUT03 cDNA library according to the recommended protocols in the SuperScriptTM
plasmid system (Catalog #18248-013, GIBCO BRL, Gaithersburg, MD). cDNAs were fractionated on a Sepharose CL4B column (Catalog #275105, Pharmacia Amersham Biotech, Piscataway, NJ), and those cDNAs exceeding 400 by were ligated into pSport I
(Catalog #15382-013, G~BCO BRL). The recombinant plasmids were subsequently transformed into DHSaTM competent cells (Catalog #18258-012, G~sco BRL).
II. Isolation and Sequencing of cDNA Clones Plasmid DNA was released from the cells and purified using the REAL Prep 96 plasmid kit (Catalog #26173, QIAGEN Inc). The recommended protocol was employed except for the following changes: 1 ) the bacteria were cultured in 1 ml of sterile Terrific Broth (Catalog #22711, G~aco BRL) with carbenicillin at 25 mg/1 and glycerol at 0.4%; 2) ~o after the cultures were incubated for 19 hours, the cells were lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid DNA pellets were each resuspended in 0.1 ml of distilled water. The DNA samples were stored at 4°C.
The cDNAs were sequenced by the method of Sanger et al. ( 1975, J. Mol. Biol.
94:441 f), using a Hamilton Micro Lab 2200 (Hamilton, Reno, NV) in combination with I5 Pettier Thermal Cyclers (PTC200 from MJ Research, Watertown, MA) and Applied Biosystems 377 DNA Sequencing Systems. and the reading frame was determined.
III. Similarity Searching of cDNA Clones and Their Deduced Proteins The nucleotide sequences and/or amino acid sequences of the Sequence Listing 2o 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.. Altschui, S.F. (1993) J. Mol. Evol 36:290-300; and Altschul et al. (1990) J. Mol. Biol. 215:403-410.) 25 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 3o 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).
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-'-5 for nucleotides and 10-8 for peptides.
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.
to 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 ~ 5 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 20 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.
25 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
30 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 x 10'' 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 x 10'3 or less are considered in the functional analyses of the protein sequences in the Sequence Listing.
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.) The PFAM database contains protein sequences of 527 protein families gathered 1o 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'°°-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 ?o or comparison sequences.
Two other algorithms, SIGPEPT and TM, both based on the HMM algorithm described above (see, e.g., Eddy, s. upra; and Sonnhammer, su ra), 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 3o 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 1 I 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.
IV. Northern Analysis 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 1o 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.) Analogous computer techniques applying BLAST are used to search for identical or related molecules in nucleotide databases such as GenBank or LIFESEQTM
database (Incyte Pharmaceuticals). This analysis is much faster than multiple membrane-based t 5 hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar.
The basis of the search is the product score, which is defined as:
sequence identity x % maximum BLAST score 20 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.
25 The results of Northern analysis are reported as a list of libraries in which the transcript encoding CALREP 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.
V. Extension of CALREP Encoding Poiynucleotides The nucleic acid sequence of Incyte Clone 2109176 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 OLIGOTM 4.06 (National Biosciences, Plymouth, MN), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC
I o content of about 50% or more, and to anneal to the target sequence at temperatures of about 68°C to about 72°C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries (Gtsco BRL) were used to extend the sequence.
If more than one extension is necessary or desired, additional sets of primers are designed I s to further extend the known region.
High fidelity amplification was obtained by following the instructions for the XL-PCRTM kit (Perkin Elmer) and thoroughly mixing the enzyme and reaction mix.
PCR was performed using the Peltier Thermal Cycler (PTC200; M.J. Research. Watertown.
MA), beginning with 40 pmol of each~primer and the recommended concentrations of all other 2o components of the kit, with the following parameters:
Step 1 94 C for 1 min (initial denaturation) Step 2 65 C for 1 min Step 3 68 C for 6 min Step 4 94 C for 15 sec 2s Step 5 65 C for 1 min Step 6 68 C for 7 min Step 7 Repeat steps 4 through 6 for an additional 15 cycles Step 8 94 C for 15 sec Step 9 65 C for 1 min 3o Step 10 68 C for 7:15 min Step 11 Repeat steps 8 through 10 for an additional 12 cycles Step 12 72 C for 8 min Step 13 4 C (and holding) 35 A S ,ul to 10 ~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 _97_ were successful in extending the sequence. Bands thought to contain the largest products were excised from the gel, purified using QIAQUICKTM (QIAGEN Inc.), and trimmed of overhangs using Klenow enzyme to facilitate religation and cloning.
After ethanol precipitation, the products were redissolved in 13 ~1 of ligation buffer, l,ul T4-DNA ligase (15 units) and l,ul T4 polynucleotide kinase were added, and the mixture was incubated at room temperature for 2 to 3 hours, or overnight at 16° C.
Competent E. coli cells (in 40 ~1 of appropriate media) were transformed with 3 ~1 of ligation mixture and cultured in 80 ~1 of SOC medium. (See, e.g., Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37°C, the E. coli mixture was plated to on Luria Bertani (LB) agar (See, e.g., Sambrook, supra, Appendix A, p. 1) containing carbenicillin (2x carb). The following day, several colonies were randomly picked from each plate and cultured in 150 ul of liquid LB/2x Garb medium placed in an individual well of an appropriate commercially-available sterile 96-well microtiter plate. The following day, ~ ul of each overnight culture was transferred into a non-sterile 96-well plate and, after dilution 1:10 with water, 5 ul from each sample was transferred into a PCR
array.
For PCR amplification. 18 ,ul of concentrated PCR reaction mix (3.3x) 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 2o performed using the following conditions:
Step 1 94 C for 60 sec Step 2 94 C for 20 sec Step 3 SS C for 30 sec Step 4 72 C for 90 sec Step S Repeat steps 2 through 4 for an additional 29 cycles Step 6 72 C for 180 sec Step 7 4 C (and holding) Aliquots of the PCR reactions were run on agarose gels together with molecular 3o 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.
In like manner, the nucleotide sequence of SEQ ID N0:2 is used to obtain 5' regulatory sequences using the procedure above. oligonucleotides designed for 5' extension, and an appropriate genomic library.
_48_ VI. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID N0:2 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 OLIGOT'" 4.06 software (National Biosciences) and labeled by combining SO
pmol of each oligomer, 250 ~Ci of [y-''-P] adenosine triphosphate (Amersham, Chicago, IL), and T4 polynucleotide kinase (DuPont NEN~, Boston, MA). The labeled oligonucleotides are substantially purified using a SephadexTM G-25 superfine size exclusion dextran bead column (Pharmacia & Upjohn. Kalamazoo, MI). An aliquot containing 10' counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xbal, or Pvu II (DuPont NEN, Boston, MA).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham, NH).
Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT ARTM film (Kodak, Rochester, NY) is exposed to the blots to film for several hours, hybridization patterns are 3o compared visually.
VII. Microarrays 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 35 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 30 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.
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 LASERGENETM. 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 Shalom 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.
VIII. Complementary Polynucleotides Sequences complementary to the CALREP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring CALREP.
~5 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 OLIGOT"' 4.06 software and the coding sequence of CALREP. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the 2o coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the CALREP-encoding transcript.
IX. Expression of CALREP
Expression and purification of CALREP is achieved using bacterial or virus-based 25 expression systems. For expression of CALREP 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 TS or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are 30 transformed into suitable bacterial hosts, e.g., BL21(DE3). Antibiotic resistant bacteria express CALREP upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG).
Expression of CALREP 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 CALREP 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 (Sf~) 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.
to Gene Ther. 7:1937-1945.) In most expression systems, CALREP 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, t5 enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Pharmacia, Piscataway, NJ).
Following purification, the GST moiety can be proteolytically cleaved from CALREP at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman 2o Kodak. Rochester, NY). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN Inc, Chatsworth, CA). Methods for protein expression and purification are discussed in Ausubeh F. M. et al. ( 1995 and periodic supplements) Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York, NY, ch 10, 16. Purified CALREP obtained by these methods can be used directly in the 25 following activity assay.
X. Demonstration of CALREP Activity An assay for CALREP activity measures the binding of CALREP to Ca'-' using a Ca'-' overlay system. (Weis, K. et al. (1994) J. Biol. Chem. 269:19142-19150.) Purified 3o CALREP is transferred and immobilized onto a nitrocellulose membrane. The membrane is washed three times with buffer (60 mM KC1, 5 mM MgCI,, 10 mM imidazole-HCI, pH
6.8) and incubated in this buffer for 10 minutes with 1 pCi [~SCa'-~] (NEN-DuPont, Boston, -sl-MA). Unbound [45Ca'-+] is removed from the membrane by washing with water, and the membrane is dried. Membrane-bound [~SCa'--J is detected by autoradiography and quantified using image analysis systems and software. CALREP activity is proportional to the amount of [~SCa'-~] detected on the membrane.
XI. Functional Assays CALREP function is also assessed by expressing the sequences encoding CALREP at physiologically elevated levels in mammalian cell culture systems.
cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives to high levels of cDNA expression. Vectors of choice include pCMV SPORTTM
(Life Technologies, Gaithersburg, MD) and pCRTM 3.1 (Invitrogen, Carlsbad, CA, both of which contain the cytomegalovirus promoter. 5-10 ~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 ~,g of an additional t 5 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, CA), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated.
laser 20 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 25 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 3o discussed in Ormerod, M. G. (1994) Flow Cytometry, Oxford, New York, NY.
The influence of CALREP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding CALREP 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, NY). mRNA can be purified from the cells using methods well known by those of skill in the art.
Expression of mRNA encoding CALREP and other genes of interest can be analyzed by Northern analysis or microarray techniques.
XII. Production of CALREP Specific Antibodies 1o CALREP substantially purified using polyacrylamide gel electrophoresis (PAGE)(see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the CALREP amino acid sequence is analyzed using ~ 5 LASERGENETM software (DNASTAR Inc.) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel supra, ch. 11.) 2o Typically, oligopeptides 1 S residues in length are synthesized using an Applied Biosystems Peptide Synthesizer Model 431 A 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 su ra.) Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant.
Resulting 25 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.
XIII. Purification of Naturally Occurring CALREP Using Specific Antibodies 3o Naturally occurring or recombinant CALREP is substantially purified by immunoaffinity chromatography using antibodies specific for CALREP. An imrnunoaffinity column is constructed by covalently coupling anti-CALREP
antibody to an activated chromatographic resin, such as CNBr-activated Sepharose (Pharmacia &
Upjohn). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing CALREP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of CALREP
(e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/CALREP 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 CALREP is collected.
1o XIV. Identification of Molecules Which Interact with CALREP
CALREP, or biologically active fragments thereof, are labeled with''-SI
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 CALREP, washed, and any wells with labeled CALREP complex are assayed.
Data obtained using different concentrations of CALREP are used to calculate values for the number, affinity. and association of CALREP with the candidate molecules.
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 2o 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 LISTING
<110> INCYTE PHARMACEUTICALS, INC.
TANG, Y. Tom CORLEY, Neil C.
GUEGLER, Karl J.
PATTERSON, Chandra <120> CALMODULIN RELATED PROTEIN
<130> PF-0528 PCT
<140> To Be Assigned <141> Herewith <150> 09/083,908 <151> 1998-05-22 <160> 3 <170> Perl Program <210> 1 <2I1> 386 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte clone 2109176 <400> 1 Met Tyr Gly Glu Cys Arg Thr Tyr Ile Ile His Tyr Tyr Leu Met Asp Asp Thr Val Glu Ile Arg Glu Val His Glu Arg Asn Asp Gly Arg Asp Pro Phe Pro Leu Leu Met Asn Arg Gln Arg Val Pro Lys Val Leu Val Glu Asn Ala Lys Asn Phe Pro Gln Cys Val Leu Glu Ile Ser Asp Gln Glu Val Leu Glu Trp Tyr Thr Ala Lys Asp Phe Ile Val Gly Lys Ser Leu Thr Ile Leu Gly Arg Thr Phe Phe Ile Tyr Asp Cys Asp Pro Phe Thr Arg Arg Tyr Tyr Lys Glu Lys Phe Gly Ile Thr Asp Leu Pro Arg Ile Asp Val Ser Lys Arg Glu Pro Pro Pro Val Lys Gln Glu Leu Pro Pro Tyr Asn Gly Phe Gly Leu Val Glu Asp Ser Ala Gln Asn Cys Phe Ala Leu Ile Pro Lys Ala Pro Lys Lys Asp Val Ile Lys Met Leu Val Asn Asp Asn Lys Val Leu Arg Tyr Leu Ala Val Leu Glu Ser Pro Ile Pro Glu Asp Lys Asp Arg Arg Phe Val Phe Ser Tyr Phe Leu Ala Thr Asp Met Ile Ser Ile Phe Glu Pro Pro Val Arg Asn Ser Gly Ile Ile Gly Gly Lys Tyr Leu Gly Arg Thr Lys Val Val Lys Pro Tyr Ser Thr Val Asp Asn Pro Val Tyr Tyr Gly Pro Ser Asp Phe Phe Ile Gly Ala Val Ile Glu Val Phe Gly His Arg Phe Ile Ile Leu Asp Thr Asp Glu Tyr Val Leu Lys Tyr Met Glu Ser Asn Ala Ala Gln Tyr Ser Pro Glu Ala Leu Ala Ser Ile Gln Asn His Val Arg Lys Arg Glu Ala Pro Ala Pro Glu Ala Glu Ser Lys Gln Thr Glu Lys Asp Pro Gly Val Gln Glu Leu Glu Ala Leu Ile Asp Thr Ile Gln Lys Gln Leu Lys Asp His Ser Cys Lys Asp Asn Ile Arg Glu Ala Phe Gln Ile Tyr Asp Lys Glu Ala Ser Gly Tyr Val Asp Arg Asp Met Phe Phe Lys Ile Cys Glu Ser Leu Asn Val Pro Val Asp Asp Ser Leu Val Lys Glu Leu Ile Arg Met Cys Ser His Gly Glu Gly Lys Ile Asn Tyr Tyr Asn Phe Val Arg Ala Phe Ser Asn <210> 2 <211> 1338 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte clone 2109176 <400> 2 cccatcagac tttgatcaac tcaagcaatt tctcaccttt gacaaacagg tccttcgatt 60 ctatgcaatc tgggatgata cagacagcat gtatggtgaa tgtcggacct acatcattca 120 ttactatctt atggatgata cggtggaaat tcgagaggtc cacgaacgga atgatgggag 180 agatcctttc ccactcctaa tgaaccgcca gcgtgtgccc aaagttttgg tggaaaatgc 240 aaagaacttc cctcagtgtg tgctagaaat ctctgaccaa gaagtgttgg aatggtatac 300 tgctaaagac ttcattgttg ggaagtcact cactatcctt gggagaactt tcttcattta 360 tgattgtgat ccatttactc gacggtatta caaagagaag tttggaatca ctgatttacc 420 acgtattgat gtgagcaagc gggaaccacc tccagtaaaa caggagttgc ctccttataa 480 cggttttgga ctagtggaag attctgctca gaattgtttt gctctcattc caaaagctcc 540 aaaaaaagac gttattaaaa tgctggtgaa tgataacaag gtgcttcgtt atttggctgt 600 actggaatcc cccatcccag aagacaaaga ccgcagattt gtcttctctt actttctagc 660 taccgacatg atcagtatct ttgagcctcc tgttcgcaat tctggtatca ttgggggcaa 720 gtaccttggc aggactaaag ttgttaaacc atactctaca gtggacaacc ctgtctacta 780 tggccccagt gacttcttca ttggtgctgt gattgaagtg tttggtcacc ggttcatcat 840 ccttgataca gacgagtatg ttttgaaata catggagagc aacgctgccc agtattcacc 900 2l3 agaagcactc gcgtcaattc agaaccatgt ccgaaagcga gaagcgcctg ctccagaagc 960 agaaagcaag caaactgaaa aggatccagg cgtgcaggaa ttggaagcat taatagacac 1020 aattcagaag caactgaaag atcactcatg caaagacaac attcgtgagg catttcaaat 1080 ttatgacaag gaagcttcag gatatgtgga cagagacatg ttctttaaaa tctgtgaatc 1140 gcttaacgtc ccagtggatg actccttggt taaggagtta atcaggatgt gctctcatgg 1200 agaaggcaaa attaactact ataactttgt tcgtgctttc tcaaactgac ctgctgatga 1260 gaaaatgcaa gacaattttt gatactggaa ctatgctttg aaatacacct tacactcttc 1320 atagaggcaa aaaaaaaa 1338 <210> 3 <211> 149 <212> PRT
<213> Homo sapiens <300>
<308> 8189081 <400> 3 Met Ala Asp Gln Leu Thr Glu Glu Gln Val Thr Glu Phe Lys Glu Ala Phe Ser Leu Phe Asp Lys Asp Gly Asp Gly Cys Ile Thr Thr Arg Glu Leu Gly Thr Val Met Arg Ser Leu Gly Gln Asn Pro Thr Glu Ala Glu Leu Arg Asp Met Met Ser Glu Ile Asp Arg Asp Gly Asn Gly Thr Val Asp Phe Pro Glu Phe Leu Gly Met Met Ala Arg Lys Met Lys Asp Thr Asp Asn Glu Glu Glu Ile Arg Glu Ala Phe Arg Val Phe Asp Lys Asp Gly Asn Gly Phe Val Ser Ala Ala Glu Leu Arg His Val Met Thr Arg Leu Gly Glu Lys Leu Ser Asp Glu Glu Val Asp Glu Met Ile Arg Ala Ala Asp Thr Asp Gly Asp Gly Gln Val Asn Tyr Glu Glu Phe Val Arg Val Leu Val Ser Lys
Claims (22)
1. A substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
2. A substantially purified variant having at least 90% amino acid sequence identity to the sequence of claim 1.
3. An isolated and purified polynucleotide encoding the polypeptide of claim 1.
4. An isolated and purified polynucleotide variant having at least 90%
polynucleotide sequence identity to the polynucleotide of claim 3.
polynucleotide sequence identity to the polynucleotide of claim 3.
5. An isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide of claim 3.
6. An isolated and purified polynucleotide which is complementary to the polynucleotide of claim 3.
7. An isolated and purified polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2.
8. An isolated and purified polynucleotide variant having at least 90%
polynucleotide sequence identity to the polynucleotide of claim 7.
polynucleotide sequence identity to the polynucleotide of claim 7.
9. An isolated and purified polynucleotide having a sequence complementary to the polynucleotide of claim 7.
10. An expression vector comprising at least a fragment of the polynucleotide of claim 3.
11. A host cell comprising the expression vector of claim 10.
12. A method for producing a polypeptide comprising a 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 of claim 11 under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
NO:1 or a fragment of SEQ ID NO:1, the method comprising the steps of:
(a) culturing the host cell of claim 11 under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
13. A pharmaceutical composition comprising the polypeptide of claim 1 in conjunction with a suitable pharmaceutical carrier.
14. A purified antibody which specifically binds to the polypeptide of claim 1.
15. A purified agonist of the polypeptide of claim 1.
16. A purified antagonist of the polypeptide of claim 1.
17. A method for treating or preventing a cell proliferative disorder, the method comprising administering to a subject in need of such treatment an effective amount of the pharmaceutical composition of claim 13.
18. A method for treating or preventing a cell proliferative disorder, the method comprising administering to a subject in need of such treatment an effective amount of the antagonist of claim 16.
19. A method for treating or preventing a neurological disorder, the method comprising administering to a subject in need of such treatment an effective amount of the pharmaceutical composition of claim 13.
20. A method for treating or preventing a neurological disorder, the method comprising administering to a subject in need of such treatment an effective amount of the antagonist of claim 16.
21. A method for detecting a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 in a biological sample containing nucleic acids, the method comprising the steps of:
(a) hybridizing the polynucleotide of claim 6 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 in the biological sample.
(a) hybridizing the polynucleotide of claim 6 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 in the biological sample.
22. The method of claim 21 wherein the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US8390898A | 1998-05-22 | 1998-05-22 | |
US09/083,908 | 1998-05-22 | ||
PCT/US1999/010747 WO1999061609A1 (en) | 1998-05-22 | 1999-05-14 | Calmodulin related protein |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2333006A1 true CA2333006A1 (en) | 1999-12-02 |
Family
ID=22181433
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002333006A Abandoned CA2333006A1 (en) | 1998-05-22 | 1999-05-14 | Calmodulin related protein |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1080190A1 (en) |
JP (1) | JP2002516097A (en) |
AU (1) | AU4080199A (en) |
CA (1) | CA2333006A1 (en) |
WO (1) | WO1999061609A1 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2377635B (en) | 2001-04-17 | 2005-10-19 | Hoffmann La Roche | Novel use of neuronal calcium sensor-1 (NCS-1) in therapy of CNS disorders and in the development of therapeutic agents |
JP5673641B2 (en) * | 2006-09-26 | 2015-02-18 | 東レ株式会社 | Immune inducer and its use |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0731166A3 (en) * | 1995-03-06 | 1997-12-29 | Tonen Corporation | Novel calcium-binding proteins |
WO1997034013A1 (en) * | 1996-03-11 | 1997-09-18 | Human Genome Sciences, Inc. | Chemotactic cytokine ii |
-
1999
- 1999-05-14 AU AU40801/99A patent/AU4080199A/en not_active Abandoned
- 1999-05-14 EP EP99924255A patent/EP1080190A1/en not_active Withdrawn
- 1999-05-14 WO PCT/US1999/010747 patent/WO1999061609A1/en not_active Application Discontinuation
- 1999-05-14 JP JP2000550993A patent/JP2002516097A/en active Pending
- 1999-05-14 CA CA002333006A patent/CA2333006A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
AU4080199A (en) | 1999-12-13 |
JP2002516097A (en) | 2002-06-04 |
WO1999061609A1 (en) | 1999-12-02 |
EP1080190A1 (en) | 2001-03-07 |
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