WO1999064593A2 - Proteines de regulation du cycle cellulaire - Google Patents

Proteines de regulation du cycle cellulaire Download PDF

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Publication number
WO1999064593A2
WO1999064593A2 PCT/US1999/012906 US9912906W WO9964593A2 WO 1999064593 A2 WO1999064593 A2 WO 1999064593A2 US 9912906 W US9912906 W US 9912906W WO 9964593 A2 WO9964593 A2 WO 9964593A2
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cecrp
seq
polynucleotide
polypeptide
sequence
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PCT/US1999/012906
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English (en)
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WO1999064593A3 (fr
Inventor
Olga Bandman
Preeti Lal
Y. Tom Tang
Neil C. Corley
Karl J. Guegler
Mariah R. Baughn
Chandra Patterson
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Incyte Pharmaceuticals, Inc.
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Priority to EP99933509A priority Critical patent/EP1086222A2/fr
Priority to CA002329688A priority patent/CA2329688A1/fr
Priority to JP2000553583A priority patent/JP2002517243A/ja
Priority to AU49554/99A priority patent/AU4955499A/en
Publication of WO1999064593A2 publication Critical patent/WO1999064593A2/fr
Publication of WO1999064593A3 publication Critical patent/WO1999064593A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to nucleic acid and amino acid sequences of cell cycle regulation proteins and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative and immune disorders.
  • Cell division is the fundamental process by which all living things grow and reproduce. In unicellular organisms such as yeast and bacteria, each cell division doubles the number of organisms, while in multicellular species many rounds of cell division are required to replace cells lost by wear or by programmed cell death, and for cell differentiation to produce a new tissue or organ. Details of the cell division cycle may vary, but the basic process consists of three principle events. The first event, interphase, involves preparations for cell division, replication of the DNA, and production of essential proteins. In the second event, mitosis, the nuclear material is divided and separates to opposite sides of the cell. The final event, cytokinesis, is division and fission of the cell cytoplasm. The sequence and timing of cell cycle transitions is under the control of the cell cycle regulation system which controls the process by positive or negative regulatory circuits at various check points.
  • Regulated progression of the cell cycle depends on the integration of growth control pathways with the basic cell cycle machinery.
  • Cell cycle regulators have been identified by selecting for human and yeast cDNAs that block or activate cell cycle arrest signals in the yeast mating pheromone pathway when they are over expressed.
  • Known regulators include human CPR (cell cycle progression restoration) genes, such as CPR8 and CPR2, and yeast CDC (cell division control) genes, including CDC91, that block the arrest signals.
  • the CPR genes express a variety of proteins including cyclins, tumor suppressor binding proteins, chaperones, transcription factors, translation factors, and RNA-binding proteins (Edwards, M. C.
  • Cdks cyclin-dependent kinases
  • the Cdks are composed of a kinase subunit, Cdk, and an activating subunit, cyclin, in a complex that is subject to many levels of regulation. There appears to be a single Cdk in S. cerevisiae and S. pombe whereas mammals have a variety of specialized Cdks. Cyclins act by binding to and activating cyclin-dependent protein kinases which then phosphorylate and activate selected proteins involved in the mitotic process.
  • Cdks cyclin-dependent kinases
  • Cdk-cyclin complex is both positively and negatively regulated by phosphorylation, and by targeted degradation involving molecules such as CDC4 and CDC53.
  • Cdks are further regulated by binding to inhibitors and other proteins such as Sucl that modify their specificity or accessibility to regulators. (Patra, D., and Dunphy, W. G. (1996) Genes Dev. 10:1503-1515; and Mathias, N. et al. (1996) Mol. Cell Biol. 16: 6634- 6643.)
  • mRNA processing includes differential, or alternate splicing of exons, which allows the expression of functionally distinct proteins from similar mRNAs. Both constitutive and alternative splicing occurs on spliceosomes, which are complex particles composed of small nuclear ribonucleoproteins (snRNPs) and non-snRNP proteins.
  • spliceosomes which are complex particles composed of small nuclear ribonucleoproteins (snRNPs) and non-snRNP proteins.
  • spliceosomes which are complex particles composed of small nuclear ribonucleoproteins (snRNPs) and non-snRNP proteins.
  • the SR (serine- and arginine-rich) family of non-snRNP splicing factors are required at early stages of spliceosome assembly, have distinct but overlapping specificities for different pre-mRNAs, and can alter splice site choice.
  • HRS a mammalian SR protein
  • ASF/SF2 a mammalian SR protein
  • SC-35 Several of the mammalian SR proteins including HRS, ASF/SF2, and SC-35, have been characterized.
  • the sequence of HRS is related to the human ASF/SF2 protein and the Drosophila B52/SRp55 protein. The latter has been shown to function in vitro to regulate 5-prime splice site selection. (Diamond, R. H. et al. (1993) J. Biol. Chem. 268:15185-15192.) Cell cycle progression also requires coordinated expression of other proteins.
  • Blimp- 1 regulatory factor appears during the differentiation of B lymphocytes into immunoglobulin secretory cells.
  • Blimp- 1 contains Kruppel-type zinc finger motifs and proline-rich and acidic regions similar to those of known transcription factors. Stable or transient transfection of Blimp- 1 into B cell lymphoma lines leads to the expression of many of the phenotypic changes associated with B cell differentiation into an early plasma cell stage, including induction of J chain message and immunoglobulin secretion, up-regulation of syndecan-1, and increased cell size and granularity. Inhibin, a member of the transforming growth factor beta-like family has been shown to modulate cell differentiation and proliferation by antagonizing the DNA synthesis activity of activin. (Turner, CA. et al. (1994) Cell (1994) 77:297-306; and Matzuk, M. M. et al. (1994) Proc. Nat. Acad. Sci. 91 :8817-8821.)
  • Progression through the cell cycle and consequently, cell proliferation is governed by the complex interactions of protein complexes composed of cyclins, cyclin-dependent protein kinases, cell growth factors, binding of inhibitory polypeptides, and associated proteins.
  • Cancers and immune disorders are characterized by uncoordinated cell proliferation. Certain cell proliferation disorders can be identified by changes in the protein complexes that normally control progression through the cell cycle.
  • a primary treatment strategy involves reestablishing control over cell cycle progression by manipulation of the proteins involved in cell cycle regulation. (Nigg, E.A.
  • the invention features substantially purified polypeptides, cell cycle regulation proteins, referred to collectively as “CECRP” and individually as “CECRP-1", “CECRP-2”, “CECRP-3”, “CECRP-4”, and “CECRP-5”.
  • CECRP substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, (SEQ ID NO:l through 5) and fragments thereof.
  • the invention further provides a substantially purified variant having at least 90% amino acid identity to the amino acid sequences of SEQ ID NO: 1 through 5, and fragments thereof.
  • the invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l through 5, and fragments thereof.
  • the invention also includes an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 through 5, and fragments thereof.
  • the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting SEQ ID NO:l through 5, and fragments thereof, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:l through 5, and fragments thereof.
  • the invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, (SEQ ID NO:6 through 10), and fragments thereof.
  • the invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:6 through 10, and fragments thereof as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 6 through 10, and fragments thereof.
  • the invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 through 5, and fragments thereof.
  • the expression vector is contained within a host cell.
  • the invention also provides a method for producing a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:l through 5, and fragments thereof, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide 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
  • a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 1 through 5, and fragments thereof in conjunction with a suitable pharmaceutical carrier.
  • the invention further includes a purified antibody which binds to a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:l through 5, and fragments thereof, as well as a purified agonist and a purified antagonist to the polypeptide.
  • the invention also provides 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 a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:l through 5, and fragments thereof in conjunction with a suitable pharmaceutical carrier.
  • the invention also provides 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 an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 through 5 and fragments thereof.
  • the invention also provides a method for treating or preventing an immune disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 1 through 5, and fragments thereof in conjunction with a suitable pharmaceutical carrier.
  • the invention also provides a method for treating or preventing an immune disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 through 5, and fragments thereof.
  • the invention also provides a method for detecting a polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:l through 5, and fragments thereof in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide sequence encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:l through 5, and fragments thereof 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.
  • the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.
  • CECRP refers to the amino acid sequences of substantially purified CECRP 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.
  • agonist refers to a molecule which, when bound to CECRP, increases or prolongs the duration of the effect of CECRP.
  • Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of CECRP.
  • allelic variant is an alternative form of the gene encoding CECRP. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • altered nucleic acid sequences encoding CECRP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide the same as CECRP or a polypeptide with at least one functional characteristic of CECRP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular ohgonucleotide probe of the polynucleotide encoding CECRP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding CECRP.
  • 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 CECRP.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of CECRP is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and arginine
  • 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.
  • 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.
  • fragments or “antigenic fragments” refer to fragments of CECRP which are preferably about 5 to about 15 amino acids in length, most preferably 14 amino acids, and which retain some biological activity or immunological activity of CECRP.
  • amino acid sequence is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • Amplification 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.)
  • PCR polymerase chain reaction
  • Antagonist refers to a molecule which, when bound to CECRP, decreases the amount or the duration of the effect of the biological or immunological activity of CECRP.
  • Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules which decrease the effect of CECRP.
  • antibody refers to intact molecules as well as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding the epitopic determinant.
  • Antibodies that bind CECRP 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 can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired.
  • a carrier protein e.g., bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH).
  • KLH keyhole limpet hemocyanin
  • the coupled peptide is then used to immunize the animal.
  • antigenic determinant refers to that fragment of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • antisense 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.
  • biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
  • immunologically active refers to the capability of the natural, recombinant, or synthetic CECRP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • complementarity refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base pairing.
  • sequence A-G-T
  • complementary sequence T-C-A
  • Complementarity between two single-stranded molecules may be "partial,” such that only some of the nucleic acids bind, or it may be “complete,” such that total complementarity exists between the single stranded molecules.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands, and in the design and use of peptide nucleic acid (PNA) molecules.
  • PNA peptide nucleic acid
  • composition comprising a given polynucleotide sequence or a “composition comprising a given amino acid sequence,” as these terms are used herein, refer broadly to any composition containing the given polynucleotide or amino acid sequence.
  • the composition may comprise a dry formulation, an aqueous solution, or a sterile composition.
  • Compositions comprising polynucleotide sequences encoding CECRP or fragments of CECRP may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • 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.
  • salts e.g., NaCI
  • detergents e.g., sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Consensus sequence 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 GEL VIEWTM Fragment Assembly system (GCG, Madison, WI). Some sequences have been both extended and assembled to produce the consensus sequence.
  • 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 CECRP, by Northern analysis is indicative of the presence of nucleic acids encoding CECRP in a sample, and thereby correlates with expression of the transcript from the polynucleotide encoding CECRP.
  • 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.
  • derivative refers to the chemical modification of a polypeptide sequence, or a polynucleotide sequence. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • similarity refers to a degree of complementarity. There may be partial similarity or complete similarity.
  • the word "identity” may substitute for the word “similarity.”
  • a partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as “substantially similar.”
  • the inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization, and the like) under conditions of reduced stringency.
  • a substantially similar sequence or hybridization probe will compete for and inhibit the binding of a completely similar (identical) sequence to the target sequence under conditions of reduced stringency. This is not to say that conditions of reduced stringency are such that non-specific binding is permitted, as reduced stringency conditions require that the binding of two sequences to one another be a specific (i.e., a selective) interaction.
  • the absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% similarity or identity). In the absence of non-specific binding, the substantially similar sequence or probe will not hybridize to the second non-complementary target sequence.
  • a partial degree of complementarity e.g., less than about 30% similarity or identity
  • Percent identity refers 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 MegAlignTM 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 is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between nucleic acid sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol 183:626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions.
  • HACs Human artificial chromosomes
  • HACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for stable mitotic chromosome segregation and maintenance. (See, e.g., Harrington, J.J. et al. (1997) Nat Genet. 15:345-355.)
  • humanized antibody 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 refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t 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).
  • 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.
  • Immuno 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.
  • factors e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
  • microarray 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.
  • element or “array element” as used herein in a microarray context, refer to hybridizable polynucleotides arranged on the surface of a substrate.
  • modulate refers to a change in the activity of
  • modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of CECRP.
  • nucleic acid or “nucleic acid sequence,” as used herein, refer to a nucleotide, ohgonucleotide, 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.
  • PNA peptide nucleic acid
  • fragments refers to those nucleic acid sequences which, comprise a region of unique polynucleotide sequence that specifically identifies a polynucleotide of SEQ ID NO:6-10, for example, as distinct from any other sequence in the same genome.
  • a fragment of a polynucleotide of SEQ ID NO:6-10 is useful in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO:6-10 from related polynucleotide sequences.
  • a fragment of a polynucleotide of SEQ ID NO:6-10 is at least about 15-20 nucleotides in length.
  • a promoter is operably associated or operably linked with a coding sequence if the promoter controls the translation of the encoded polypeptide. While operably associated or operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements, e.g., repressor genes, are not contiguously linked to the sequence encoding the polypeptide but still bind to operator sequences that control expression of the polypeptide.
  • oligonucleotide refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray.
  • oligonucleotide is substantially equivalent to the terms “amplimer,” “primer,” “oligomer,” and “probe,” as these terms are commonly defined in the art.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition.
  • PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63.)
  • sample is used in its broadest sense.
  • a biological sample suspected of containing nucleic acids encoding CECRP, or fragments thereof, or CECRP 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.
  • 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.
  • 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.
  • stringent conditions refers to conditions which permit hybridization between polynucleotides and the claimed polynucleotides.
  • Stringent conditions can be defined by salt concentration, the concentration of organic solvent (e.g., formamide), temperature, and other conditions well known in the art.
  • stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature.
  • substantially purified 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.
  • substitution refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
  • Transformation describes a process by which exogenous DNA enters and changes a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, viral infection, electroporation, heat shock, lipofection, and particle bombardment.
  • transformed cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
  • a “variant" of CECRP 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).
  • variants when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to CECRP. This definition may also include, for example, "allelic” (as defined above), “splice,” “species,” or “polymorphic” variants.
  • a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or an absence of domains.
  • Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other.
  • a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
  • Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
  • SNPs single nucleotide polymorphisms
  • the invention is based on the discovery of new cell cycle regulation proteins (CECRP), the polynucleotides encoding CECRP, and the use of these compositions for the diagnosis, treatment, or prevention of diseases associated with cell proliferation and immune disorders.
  • CECRP cell cycle regulation proteins
  • columns 1 and 2 show the sequence identifiers for each of the amino acid and nucleic acid sequence, respectively.
  • Column 3 shows the Clone ID of the Incyte Clone in which nucleic acids encoding each CECRP were first identified, and column 4, the cDNA library of this clone.
  • Column 5 is entitled fragments, and shows the Incyte clones (and libraries) and shotgun sequences useful as fragments, for example, in hybridization technologies, and which are part of the consensus nucleotide sequence of each CECRP.
  • column 1 references the SEQ ID NO; column 2 shows the number of amino acid residues; column 3, potential phosphorylation sites; column 4, potential glycosylation sites; column 5, identifying sequences and/or structural motifs; and column 6, analytical methods used to identify the protein through sequence homologies and protein motifs.
  • the columns of Table 3 show the tissue expression of each nucleic acid sequence by Northern analysis, diseases or disorders associated with this tissue expression, and the vector into which each cDNA was cloned.
  • the invention also encompasses CECRP variants.
  • a preferred CECRP 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 CECRP amino acid sequence, and which contains at least one functional or structural characteristic of CECRP.
  • the invention also encompasses polynucleotides which encode CECRP.
  • the invention encompasses a polynucleotide sequence comprising the sequence selected from the group consisting of SEQ ID NO:6 through 10.
  • the invention also encompasses a variant of a polynucleotide sequence encoding CECRP.
  • 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 CECRP.
  • a particular aspect of the invention encompasses a variant of a nucleic acid sequence selected from the group consisting of SEQ ID NO:6 through 10 which has at least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO : 6 through SEQ ID NO : 10.
  • Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of CECRP.
  • nucleotide sequences which encode CECRP and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring CECRP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding CECRP or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
  • 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 CECRP and CECRP derivatives, or fragments thereof, entirely by synthetic chemistry.
  • the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art.
  • synthetic chemistry may be used to introduce mutations into a sequence encoding CECRP or any fragment thereof.
  • polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO:6 through 10, or a fragments thereof, under various conditions of stringency.
  • SEQ ID NO:6 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.
  • Methods for DNA sequencing and analysis are well known and generally available in the art and may be used to practice any of the embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I,
  • sequence preparation is automated with machines such as the ABI Catalyst 800 (Perkin Elmer) or a Hamilton Micro Lab 2200 (Hamilton, Reno NV) in combination with thermal cyclers (for PCR).
  • Sequencing is then carried out using either ABI 373 or 377 DNA sequencers (Perkin Elmer) or the MEGABASE capillary electrophoresis (Molecular Dynamics), and the sequences are analyzed using tools (computer programs and algorithms) which are well known in the art. (Ausubel (1997. unit 1.1); Meyers. R.A. (1995; Molecular Biology and Biotechnology. Wiley VCH, Inc, New York NY, p 856-853).
  • the nucleic acid sequences encoding CECRP 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.
  • restriction-site PCR uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector.
  • Another method inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
  • the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences.
  • a third method involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
  • PCR Methods Applic. 1 :111-119. multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
  • Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-306).
  • primers may be designed using commercially available software, such as OLIGOTM 4.06 Primer Analysis software (National Biosciences Inc., Madison, 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.
  • OLIGOTM 4.06 Primer Analysis software National Biosciences Inc., Plymouth, MN
  • 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.
  • 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., GenotyperTM and Sequence NavigatorTM, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
  • Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
  • polynucleotide sequences or fragments thereof which encode CECRP may be cloned in recombinant DNA molecules that direct expression of CECRP, 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 CECRP.
  • nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter CECRP-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.
  • oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
  • sequences encoding CECRP may be synthesized, in whole or in part, using chemical methods well known in the art.
  • CECRP itself or a fragment thereof may be synthesized using chemical methods.
  • peptide synthesis can be performed using various solid-phase techniques.
  • the nucleotide sequences encoding CECRP or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
  • these elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding CECRP. Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding CECRP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • a variety of expression vector/host systems may be utilized to contain and express sequences encoding CECRP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
  • the invention is not limited by the host cell employed.
  • cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding CECRP.
  • routine cloning, subcloning, and propagation of polynucleotide sequences encoding CECRP can be achieved using a multifunctional E. coli vector such as Bluescript® (Stratagene) or pSportlTM plasmid (GIBCO BRL).
  • Ligation of sequences encoding CECRP into the vector's multiple cloning site disrupts the lacL gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules.
  • these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
  • vectors which direct high level expression of CECRP may be used.
  • vectors containing the strong, inducible T5 or T7 bacteriophage promoter may be used.
  • Yeast expression systems may be used for production of CECRP.
  • a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
  • such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
  • Plant systems may also be used for expression of CECRP.
  • Transcription of sequences encoding CECRP 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. 3:111.)
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used.
  • promoters such as the small subunit of RUBISCO or heat shock promoters may be used.
  • constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection.
  • pathogen-mediated transfection See, e.g., Hobbs, S. or Murry, L.E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill New York. NY; pp. 191-196.
  • a number of viral -based expression systems may be utilized.
  • sequences encoding CECRP may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence.
  • Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses CECRP in host cells.
  • CECRP CECRP
  • 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.
  • HACs 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.
  • sequences encoding CECRP can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
  • the purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type. Any number of selection systems may be used to recover transformed cell lines.
  • herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes for use in tk or apr cells, respectively.
  • herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes for use in tk or apr cells, respectively.
  • antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • neo confers resistance to the aminoglycosides neomycin and G-418
  • als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
  • Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites.
  • Visible markers e.g., anthocyanins, green fluorescent proteins (GFP) (Clontech, Palo Alto, CA), ⁇ glucuronidase and its substrate ⁇ -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, CA. et al. (1995) Methods Mol. Biol. 55:121-131.)
  • marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
  • sequence encoding CECRP is inserted within a marker gene sequence
  • transformed cells containing sequences encoding CECRP can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding CECRP 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.
  • host cells that contain the nucleic acid sequence encoding CECRP and that express CECRP may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. Immunological methods for detecting and measuring the expression of CECRP 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).
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on CECRP is preferred, but a competitive binding assay may be employed.
  • 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 Immunology. Greene Pub. Associates and Wiley-Interscience, New York, NY; and Maddox, D.E. et al. (1983) J. Exp. Med. 158:1211-1216).
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding CECRP include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • the sequences encoding CECRP, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • T7, T3, or SP6 RNA polymerase
  • RNA polymerase such as T7, T3, or SP6
  • 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 CECRP 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.
  • expression vectors containing polynucleotides which encode CECRP may be designed to contain signal sequences which direct secretion of CECRP through a prokaryotic or eukaryotic cell membrane.
  • 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.
  • 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.
  • ATCC American Type Culture Collection
  • natural, modified, or recombinant nucleic acid sequences encoding CECRP may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric CECRP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of CECRP activity.
  • Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices.
  • Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
  • a fusion protein may also be engineered to contain a proteolytic cleavage site located between the CECRP encoding sequence and the heterologous protein sequence, so that CECRP may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel, F. M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology. John Wiley & Sons, New York, NY, ch 10. A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
  • synthesis of radiolabeled CECRP 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 35 S- methionine.
  • Fragments of CECRP 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 431 A Peptide Synthesizer (Perkin Elmer). Various fragments of CECRP may be synthesized separately and then combined to produce the full length molecule.
  • CECRP cell cycle regulation proteins.
  • the expression of CECRP is closely associated with cell proliferation. Therefore, in cancers or immune disorders where CECRP is an activator, or enhancer, and is promoting cell proliferation, it is desirable to decrease the expression of CECRP. In cancers or immune disorders where CECRP is an inhibitor or suppressor of cellular processes that modulate cell proliferation it is desirable to provide the protein or to increase the expression of CECRP.
  • CECRP or a fragment or derivative thereof may be administered to a subject to treat or prevent a cell proliferation disorder.
  • a disorder may include, but is not limited to, actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pan
  • a pharmaceutical composition comprising purified CECRP may be used to treat or prevent a cell proliferative disorder including, but not limited to, those listed above.
  • an agonist which is specific for CECRP may be administered to a subject to treat or prevent a cell proliferative disorder including, but not limited to, those cancers listed above.
  • a vector capable of expressing CECRP, or a fragment or a derivative thereof may be administered to a subject to treat or prevent a cell proliferative disorder including, but not limited to, those listed above.
  • CECRP or a fragment or derivative thereof may be administered to a subject to treat or prevent an immune disorder.
  • a disorder may include, but is not limited to, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture' s syndrome, gout, Graves' disease, Hashimoto's thyroiditis
  • AIDS acquired immunodeficiency syndrome
  • a pharmaceutical composition comprising purified CECRP may be used to treat or prevent an immune disorder including, but not limited to, those listed above.
  • an agonist which is specific for CECRP may be administered to a subject to treat or prevent an immune disorder including, but not limited to, those listed above.
  • a vector capable of expressing CECRP, or a fragment or a derivative thereof may be administered to a subject to treat or prevent an immune disorder including, but not limited to, those listed above.
  • CECRP is a promoter of cell proliferation
  • antagonists which decrease the expression or activity of CECRP may be administered to a subject to treat or prevent a cell proliferation disorder including, but not limited to, those listed above.
  • antibodies which specifically bind CECRP 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 CECRP.
  • a vector expressing the complement of the polynucleotide encoding CECRP may be administered to a subject to treat or prevent a cell proliferation disorder including, but not limited to, those listed above.
  • CECRP is a promoter of cell proliferation
  • antagonists which decrease the expression or activity of CECRP may be administered to a subject to treat or prevent an immune disorder including, but not limited to, those listed above.
  • a vector expressing the complement of the polynucleotide encoding CECRP may be administered to a subject to treat or prevent an immune disorder including, but not limited to, those listed above.
  • CECRP or a fragment or derivative thereof may be added to cells to stimulate cell proliferation.
  • CECRP may be added to a cell in culture or cells in vivo using delivery mechanisms such as liposomes, viral based vectors, or electroinjection for the purpose of promoting cell proliferation and tissue or organ regeneration.
  • CECRP may be added to a cell, cell line, tissue or organ culture in vitro or ex vivo to stimulate cell proliferation for use in heterologous or autologous transplantation.
  • the cell will have been preselected for its ability to fight an infection or a cancer or to correct a genetic defect in a disease such as sickle cell anemia, ⁇ thalassemia, cystic fibrosis, or Huntington's chorea.
  • an agonist which is specific for CECRP may be administered to a cell to stimulate cell proliferation, as described above.
  • a vector capable of expressing CECRP, or a fragment or a derivative thereof may be administered to a cell to stimulate cell proliferation, as described above.
  • any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • An antagonist of CECRP may be produced using methods which are generally known in the art.
  • purified CECRP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind CECRP.
  • Antibodies to CECRP 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.
  • various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with CECRP or with any fragment or oligopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • 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
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are especially preferable.
  • the oligopeptides, peptides, or fragments used to induce antibodies to CECRP have an amino acid sequence consisting of at least about 5 amino acids, and, more preferably, of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of CECRP 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 CECRP 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.
  • the hybridoma technique the human B-cell hybridoma technique
  • 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.
  • 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.
  • 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.
  • techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce CECRP-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 CECRP may also be generated.
  • 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.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)
  • immunoassays may be used for screening to identify antibodies having the desired specificity.
  • Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.
  • Such immunoassays typically involve the measurement of complex formation between CECRP and its specific antibody.
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering CECRP epitopes is preferred, but a competitive binding assay may also be employed. (Maddox, supra.)
  • the polynucleotides encoding CECRP may be used for therapeutic purposes.
  • the complement of the polynucleotide encoding CECRP may be used in situations in which it would be desirable to block the transcription of the mRNA.
  • cells may be transformed with sequences complementary to polynucleotides encoding CECRP.
  • complementary molecules or fragments may be used to modulate CECRP activity, or to achieve regulation of gene function.
  • sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding CECRP.
  • Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors to express nucleic acid sequences complementary to the polynucleotides encoding CECRP. (See, e.g., Sambrook, supra; and Ausubel, supra.)
  • Genes encoding CECRP can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide, or fragment thereof, encoding CECRP. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector, and may last even longer if appropriate replication elements are part of the vector system.
  • modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5', or regulatory regions of the gene encoding CECRP.
  • Oligonucleotides derived from the transcription initiation site e.g., between about positions -10 and +10 from the start site, are preferred.
  • inhibition can be achieved using triple helix base-pairing methodology.
  • Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E.
  • 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 cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding CECRP.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • RNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding CECRP. 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.
  • 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.
  • vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nature Biotechnology 15:462-466.)
  • any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • 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 CECRP, antibodies to CECRP, and mimetics, agonists, antagonists, or inhibitors of CECRP.
  • 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.
  • compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules
  • auxiliaries can be added, if desired.
  • Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen.
  • 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.
  • 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.
  • Push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • 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.
  • 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.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • the pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • 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.
  • the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1 mM to 50 mM histidine, 0.1 % to 2% sucrose, and 2% to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells or in animal models such as mice, rats, rabbits, dogs, or pigs.
  • An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient, for example CECRP or fragments thereof, antibodies of CECRP, and agonists, antagonists or inhibitors of CECRP, which ameliorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeutically effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics.
  • the dose ratio of therapeutic to toxic effects is the therapeutic index, and it can be expressed as the ED 50 /LD 50 ratio.
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
  • Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • antibodies which specifically bind CECRP may be used for the diagnosis of disorders characterized by expression of CECRP, or in assays to monitor patients being treated with CECRP or agonists, antagonists, or inhibitors of CECRP.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for CECRP include methods which utilize the antibody and a label to detect CECRP 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.
  • CECRP CECRP
  • ELISAs RIAs
  • FACS fluorescence-activated cell sorting
  • RIAs RIAs
  • FACS fluorescence-activated cell sorting
  • normal or standard values for CECRP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to CECRP under conditions suitable for complex formation The amount of standard complex formation may be quantitated by various methods, preferably by photometric means. Quantities of CECRP 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.
  • the polynucleotides encoding CECRP may be used for diagnostic purposes.
  • the polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of CECRP may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of CECRP, and to monitor regulation of CECRP levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding CECRP or closely related molecules may be used to identify nucleic acid sequences which encode CECRP.
  • the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low), will determine whether the probe identifies only naturally occurring sequences encoding CECRP, 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 CECRP encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequences of SEQ ID NO: 6 through SEQ ID NO: 10 or from genomic sequences including promoters, enhancers, and introns of the CECRP gene.
  • Means for producing specific hybridization probes for DNAs encoding CECRP include the cloning of polynucleotide sequences encoding CECRP or CECRP derivatives into vectors for the production of mRNA probes.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotide sequences encoding CECRP may be used for the diagnosis of a disorder associated with expression of CECRP.
  • a disorder associated with expression of CECRP include, but are not limited to, actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancre
  • the polynucleotide sequences encoding CECRP 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 CECRP expression. Such qualitative or quantitative methods are well known in the art.
  • the nucleotide sequences encoding CECRP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding CECRP may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding CECRP 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.
  • 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 CECRP, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
  • 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.
  • oligonucleotides designed from the sequences encoding CECRP 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 CECRP, or a fragment of a polynucleotide complementary to the polynucleotide encoding CECRP, 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 CECRP include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves.
  • radiolabeling or biotinylating nucleotides include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves.
  • 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.
  • oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as targets in a microarray.
  • the microarray can be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.
  • Microarrays may be prepared, used, and analyzed using methods known in the art.
  • 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 WO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.
  • nucleic acid sequences encoding CECRP may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence.
  • the sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial chromosome cDNA libraries.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDNA libraries.
  • Fluorescent in situ hybridization may be correlated with other physical chromosome mapping techniques and genetic map data.
  • FISH Fluorescent in situ hybridization
  • 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 CECRP 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 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.
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • 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.
  • CECRP 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 CECRP and the agent 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. (1984) PCT application WO84/03564.) In this method, large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • test compounds are reacted with CECRP, or fragments thereof, and washed. Bound CECRP is then detected by methods well known in the art.
  • Purified CECRP can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • competitive drug screening assays in which neutralizing antibodies capable of binding CECRP specifically compete with a test compound for binding CECRP. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with CECRP.
  • nucleotide sequences which encode CECRP may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
  • RNA was purchased from Clontech (Palo Alto, CA) or isolated at Incyte from tissues described in Table 4. The tissue was homogenized and lysed in guanidinium isothiocyanate, and the lysate was centrifuged over a CsCl cushion. Alternatively, the tissue was homogenized and lysed in phenol or a suitable mixture of denaturants such as TRIzol reagent (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate, and the lysate was extracted with chloroform (1 :5 v/v). RNA was precipitated from lysates with either isopropanol or sodium acetate and ethanol.
  • TRIzol reagent Life Technologies
  • UNIZAP vector Stratagene, La Jolla, CA
  • Superscript plasmid system Life Technologies, Inc
  • cDNA libraries were constructed by Stratagene using RNA provided by Incyte. Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and cDNA was digested with an appropriate restriction enzyme(s).
  • cDNA was size-selected (300-1000 bp) using Sephacryl S 1000 or Sepharose CL2B or CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis.
  • cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., pBluescript (Stratagene), pSPORT 1 (Life Technologies), pINCY (Incyte Pharmaceuticals Inc, Palo Alto, CA).
  • pINCY was amplified in JM109 cells and purified using the QiaQuick column (QIAGEN Inc).
  • Recombinant plasmids were transformed into competent E. coli cells, e.g., XLl-Blue, XLl-BlueMRF, or SOLR (Stratagene) or DH5 ⁇ , DH10B, or ElectroMAX DH10B (Life Technologies).
  • Plasmids were recovered from host cells by in vivo excision (UniZAP vector system, Stratagene) or by cell lysis. Plasmids were purified using the MAGIC MINIPREPS DNA purification system (Promega, Madison, WI); Miniprep kit (Advanced Genetic Technologies Corporation, Gaithersburg, MD); QIAwell-8 Plasmid, QIAwell PLUS DNA, or QIAwell ULTRA DNA purification systems; or REAL Prep 96 plasmid kit (QIAGEN Inc) using the recommended protocol. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C
  • plasmid DNA was amplified from host cell lysates using direct link PCR (Rao, V.B. (1994) Anal. Biochem. 216:1-14) in a high-throughput format. Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates (Genetix Ltd, Wales UK) and concentration of amplified plasmid DNA was quantified fluorometrically using Pico Green Dye (Molecular Probes, Eugene OR) and a Fluoroscan II fluorescence scanner (Labsystems Oy, Helsinki, Finland).
  • the cDNAs were prepared for sequencing using either an ABI Catalyst 800 (Perkin Elmer) or a Hamilton Micro Lab 2200 (Hamilton, Reno, NV) in combination with Peltier Thermal Cyclers (PTC200; MJ Research, Watertown MA).
  • the cDNAs were sequenced on the ABI 373 or 377 DNA Sequencing systems (Perkin Elmer) by the method of Sanger F and A.R. Coulson (1975; J. Mol. Biol. 94:441-448) using standard ABI protocols, base calling software, and kits.
  • cDNAs were sequenced using solutions and dyes from Amersham Pharmacia Biotech. Reading frame was determined using standard methods (Ausubel, supra).
  • Table 5 summarizes the databases and tools used herein.
  • the first column of Table 5 shows the tool, program, or algorithm; the second column, the database; the third column, a brief description; and the fourth column (where applicable), scores for determining the strength of a match between two sequences (the higher the value, the more homologous).
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; and Ausubel, supra, ch. 4 and 16.)
  • 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 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 100
  • 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.
  • nucleic acid sequences of Incyte Clones 037377, 162871, 236062, 1596581, and 1853196 were used to design oligonucleotide primers for extending partial nucleotide sequences 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, Madison, 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 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 (GiBCO BRL) were used to extend the sequence.
  • 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 components of the kit, with the following parameters:
  • Step 1 94° C for 1 min (initial denaturation)
  • Step 3 68° C for 6 min
  • Step 4 94° C for 15 sec
  • 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
  • Step 11 Repeat steps 8 through 10 for an additional 12 cycles
  • coli mixture was plated 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 ⁇ l of liquid LB/2x carb medium placed in an individual well of an appropriate commercially-available sterile 96-well microtiter plate. The following day, 5 ⁇ l of each overnight culture was transferred into a non-sterile 96-well plate and, after dilution 1 :10 with water, 5 ⁇ l from each sample was transferred into a PCR array.
  • LB Luria Bertani
  • PCR amplification For PCR amplification, 18 ⁇ l 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 performed using the following conditions:
  • Step 1 94° C for 60 sec
  • Step 2 94° C for 20 sec
  • Step 3 55° C for 30 sec
  • Step 4 72° C for 90 sec
  • Step 5 Repeat steps 2 through 4 for an additional 29 cycles
  • nucleotide sequence of SEQ ID NO:6 through 10 to obtain 5' regulatory sequences using the procedure above, oligonucleotides designed for 5' extension, and an appropriate genomic library.
  • Hybridization probes derived from SEQ ID NO: 6 through 10 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 OLIGOTM 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ⁇ Ci of [ ⁇ - 32 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 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, 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.
  • 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.
  • XOMAT ARTM film Kerat, Rochester, NY
  • hybridization patterns are compared visually.
  • a chemical coupling procedure and an ink jet device can be used to synthesize array elements on the surface of a substrate.
  • An array analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical array may be produced by hand or using available methods and machines and contain any appropriate number of elements.
  • nonhybridized probes are removed and a scanner used to determine the levels and patterns of fluorescence. The degree of complementarity and the relative abundance of each probe which hybridizes to an element on the microarray may be assessed through analysis of the scanned images.
  • 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.
  • Fluorescent probes are prepared and used for hybridization to the elements on the substrate.
  • the substrate is analyzed by procedures described above.
  • Sequences complementary to the CECRP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring CECRP.
  • 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 OLIGOTM 4.06 software and the coding sequence of CECRP.
  • a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
  • To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the CECRP-encoding transcript.
  • CECRP CECRP protein kinase
  • cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
  • promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
  • Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
  • Antibiotic resistant bacteria express CECRP upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG).
  • CECRP CECRP in eukaryotic cells
  • baculovirus recombinant Autographica californica nuclear polyhedrosis virus
  • AcMNPV Autographica californica nuclear polyhedrosis virus
  • the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding CECRP by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
  • Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.
  • CECRP 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 glutathione S-transferase
  • a peptide epitope tag such as FLAG or 6-His
  • FLAG an 8-amino acid peptide
  • 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 Ausubel, F. M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology. John Wiley & Sons, New York, NY, ch 10, 16. Purified CECRP obtained by these methods can be used directly in the following activity assay.
  • CECRP activity is demonstrated by measuring the induction of terminal differentiation or cell cycle progression when CECRP is expressed at physiologically elevated levels in mammalian cell culture systems.
  • cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include pCMV SPORTTM (Life Technologies,
  • recombinant vector contains 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 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.
  • GFP Green Fluorescent Protein
  • Flow cytometry detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell cycle progression or terminal differentiation.
  • CECRP function is assessed by expressing the sequences encoding CECRP at physiologically elevated levels in mammalian cell culture systems.
  • cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include pCMV 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 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 an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP, and to evaluate properties, for example, their apoptotic state.
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M. G. (1994) Flow Cytometry. Oxford, New York, NY.
  • 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 CECRP and other genes of interest can be analyzed by Northern analysis or microarray techniques.
  • PAGE polyacrylamide gel electrophoresis
  • the CECRP amino acid sequence is analyzed using 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.
  • LASERGENETM software DNASTAR Inc.
  • 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.)
  • oligopeptides 15 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.
  • MBS N-maleimidobenzoyl-N-hydroxysuccinimide ester
  • Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide activity by, for example, binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio- iodinated goat anti-rabbit IgG.
  • Naturally occurring or recombinant CECRP is substantially purified by immunoaffinity chromatography using antibodies specific for CECRP.
  • An immunoaffinity column is constructed by covalently coupling anti-CECRP 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 CECRP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of CECRP (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/CECRP 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 CECRP is collected.
  • CECRP CECRP, or biologically active fragments thereof, are labeled with 125 I Bolton-Hunter reagent.
  • 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 CECRP, washed, and any wells with labeled CECRP complex are assayed. Data obtained using different concentrations of CECRP are used to calculate values for the number, affinity, and association of CECRP with the candidate molecules.
  • Phrap Quality-score based assembly program for shotgun sequences match > 56 score > 120

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Abstract

La présente invention concerne des protéines de régulation du cycle cellulaire humain (CECRP) et des polynucléotides identifiant et codant CECRP. L'invention concerne également des vecteurs d'expression, des cellules hôtes, des agonistes et des antagonistes. L'invention concerne enfin des méthodes de diagnostic, de traitement ou de prévention de troubles associés à l'expression de CECRP.
PCT/US1999/012906 1998-06-08 1999-06-08 Proteines de regulation du cycle cellulaire WO1999064593A2 (fr)

Priority Applications (4)

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EP99933509A EP1086222A2 (fr) 1998-06-08 1999-06-08 Proteines de regulation du cycle cellulaire
CA002329688A CA2329688A1 (fr) 1998-06-08 1999-06-08 Proteines de regulation du cycle cellulaire
JP2000553583A JP2002517243A (ja) 1998-06-08 1999-06-08 細胞サイクル調節タンパク質
AU49554/99A AU4955499A (en) 1998-06-08 1999-06-08 Cell cycle regulation proteins

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US8869598P 1998-06-08 1998-06-08
US60/088,695 1998-06-08

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1033402A1 (fr) * 1997-11-05 2000-09-06 Sumitomo Electric Industries, Ltd. PROTEINES DE FIXATION DE p16, GENE DE CES PROTEINES ET ANTICORPS CONTRE CES PROTEINES
WO2001075002A2 (fr) * 2000-03-02 2001-10-11 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, gène de regulation du cycle cellulaire humain 28, et polynucléotide codant pour ce polypeptide

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995028483A1 (fr) * 1994-04-14 1995-10-26 Cold Spring Harbor Laboratory Proteines regulatrices du cycle cellulaire et leurs utilisations

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995028483A1 (fr) * 1994-04-14 1995-10-26 Cold Spring Harbor Laboratory Proteines regulatrices du cycle cellulaire et leurs utilisations

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE EMEST20 [Online] E.M.B.L. Databases Accession Number: AA280100, 3 April 1997 (1997-04-03) NCI-CGAP: "Homo sapiens cDNA clone IMAGE:705030 5' similar to TR:G487787 G487787 ZINC FINGER PROTEIN ZNF140." XP002118250 *
DATABASE EMEST27 [Online] E.M.B.L. Databases Accession Number: AA307127, 18 April 1997 (1997-04-18) ADAMS M ET AL: "Homo sapiens cDNA 5' end similar to similar to zinc finger protein family" XP002118251 *
DATABASE EMSET 25 [Online] E.M.B.L. databases Accession Number: AA148046, 15 December 1996 (1996-12-15) HILLIER L ET AL: "Homo sapiens cDNA clone 590442 5' similar to TR:G577019 G577019 PROCKR2" XP002118252 *
TURNER C ET AL: "Blimp-1, a novel zinc finger-containing protein that can drive the maturation of B lymphocytes into immunoglobulin-secreting cells" CELL, vol. 77, no. 2, 22 April 1997 (1997-04-22), pages 297-306, XP002118249 cited in the application *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1033402A1 (fr) * 1997-11-05 2000-09-06 Sumitomo Electric Industries, Ltd. PROTEINES DE FIXATION DE p16, GENE DE CES PROTEINES ET ANTICORPS CONTRE CES PROTEINES
EP1033402A4 (fr) * 1997-11-05 2003-01-02 Sumitomo Electric Industries PROTEINES DE FIXATION DE p16, GENE DE CES PROTEINES ET ANTICORPS CONTRE CES PROTEINES
WO2001075002A2 (fr) * 2000-03-02 2001-10-11 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, gène de regulation du cycle cellulaire humain 28, et polynucléotide codant pour ce polypeptide
WO2001075002A3 (fr) * 2000-03-02 2002-01-24 Biowindow Gene Dev Inc Nouveau polypeptide, gène de regulation du cycle cellulaire humain 28, et polynucléotide codant pour ce polypeptide

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JP2002517243A (ja) 2002-06-18

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