EP1003880A2 - Proteines liees aux cyclines - Google Patents

Proteines liees aux cyclines

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Publication number
EP1003880A2
EP1003880A2 EP98930079A EP98930079A EP1003880A2 EP 1003880 A2 EP1003880 A2 EP 1003880A2 EP 98930079 A EP98930079 A EP 98930079A EP 98930079 A EP98930079 A EP 98930079A EP 1003880 A2 EP1003880 A2 EP 1003880A2
Authority
EP
European Patent Office
Prior art keywords
hcrp
sequence
polynucleotide sequence
leu
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98930079A
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German (de)
English (en)
Inventor
Jennifer L. Hillman
Neil C. Corley
Karn J. Guegler
Benjamin Graeme Cocks
Purvi Shah
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Incyte Corp
Original Assignee
Incyte Pharmaceuticals Inc
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Filing date
Publication date
Application filed by Incyte Pharmaceuticals Inc filed Critical Incyte Pharmaceuticals Inc
Publication of EP1003880A2 publication Critical patent/EP1003880A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • 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 two cyclin related proteins and to the use of these sequences in the diagnosis, prevention, and treatment of cancer, immune disorders, and developmental 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 produce a new tissue or organ and to replace cells lost by wear or by programmed cell death. 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.
  • interphase involves preparations for cell division, replication of the DNA and production of essential proteins.
  • mitosis the nuclear material is divided and separates to opposite sides of the cell.
  • cytokinesis is division and fission of the cell cytoplasm.
  • cyclin B which controls entry of the cell into mitosis
  • GI cyclin which controls events that drive the cell out of mitosis.
  • Cyclins are characterized by a large region of shared homology that is approximately 180 amino acids in length and referred to as the "cyclin box" (Chapman, D.L. and Wolgemuth, DJ. (1993) Development 118:229-40).
  • cyclins contain a sequence of amino acids in the N- terminal region of the molecule called the "destruction box"(Hunt, T. (1991) Nature 349: 100- 101). This sequence, which contains a conserved motif of RXXLXXIXN, is believed to be a recognition code that triggers ubiquitin-mediated degradation of cyclin B (see below).
  • Cyclins are degraded through the ubiquitin conjugation system (UCS), a major pathway for the degradation of cellular proteins in eukaroytic cells and in some bacteria.
  • UCS ubiquitin conjugation system
  • the UCS mediates the elimination of abnormal proteins and regulates the half-lives of important regulatory proteins that control cellular processes such as gene transcription and cell cycle progression.
  • the UCS is implicated in the degradation of mitotic cyclic kinases, oncoproteins, tumor suppressor genes such as p53, viral proteins, cell surface receptors associated with signal transduction, transcriptional regulators, and mutated or damaged proteins (Ciechanover, supra).
  • ubiquitin conjugation and protein degradation occurs in four principal steps (Jentsch, S. (1992) Annu. Rev. Genet. 26:179-207).
  • First ubiquitin (Ub) a small, heat stable protein (76 amino acids) is activated by a ubiquitin-activating enzyme (El) in an ATP dependent reaction which binds the C-terminus of Ub to the thiol group of an internal cysteine residue in El.
  • activated Ub is transferred to one of several Ub-conjugating enzymes (E2).
  • E2 Ub-conjugating enzymes
  • Different ubiquitin-dependent proteolytic pathways employ structurally similar, but distinct ubiquitin-conjugating enzymes that are associated with recognition subunits which direct them to proteins carrying a particular degradation signal.
  • E2 then links the Ub molecule through its C- terminal glycine to an internal lysine (acceptor lysine) of a target protein. Additional Ub molecules may be added forming a multi-Ub chain structure. The ubiquinated protein is then recognized and degraded by proteasome, a large, multisubunit proteolytic enzyme complex, and Ub is released for reutilization.
  • the E2 (Ub-conjugating) enzymes are important for substrate specificity in different UCS pathways. All E2s have a conserved domain of approximately 16 kDa called the UBC domain that is at least 35% identical in all E2s and contains a centrally located cysteine residue required for ubiquitin-enzyme thiolester formation (Jentsch, supra). A well conserved proline-rich element is located N-terminal to the active cysteine residue. Structural variations beyond this conserved domain are used to classify the E2 enzymes. Class I E2s consist almost exclusively of the conserved UBC domain. Class II E2s have various unrelated C-terminal extensions that contribute to substrate specificity and cellular localization.
  • Class HI E2s have unique N-terminal extensions which are believed to be involved in enzyme regulation or substrate specificity. Recently, a new type of E2 has been found that is selective for mitotic cyclins (Aris tarkhov, A.et al. (1996) Proc. Natl. Acad. Sci. 93:4294-99). Cyclin-specific E2, or E2-C, is characterized by the conserved UBC domain, an N-terminal extension of 30 amino acids not found in other E2s, and a unique sequence, TLLMSGD, adjacent to this extension. These characteristics together with the high affinity of E2-C for cyclin identify it as a new class of E2. The discovery of new cyclin related proteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of cancer, immune disorders, and developmental disorders.
  • the invention features a substantially purified polypeptide, cyclin related protein HCRP-1 having the amino acid sequence shown in SEQ ID NOJ, or fragments thereof.
  • the invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or fragments thereof and a composition comprising said polynucleotide sequence.
  • the invention also provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the amino acid sequence SEQ ID NOJ, or fragments of said polynucleotide sequence.
  • the invention further provides a polynucleotide sequence comprising the complement of the polynucleotide sequence encoding the amino acid sequence of SEQ ID NOJ, or fragments or variants of said polynucleotide sequence.
  • the invention also provides an isolated and purified sequence comprising SEQ ID NO.2 or variants thereof.
  • the invention provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:2.
  • the invention provides a composition comprising an isolated and purified polynucleotide sequence comprising the complement of SEQ ID NO:2, or fragments or variants thereof.
  • the invention also provides a polynucleotide sequence comprising the complement of SEQ ID NO:2.
  • the present invention further provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences.
  • the expression vector containing the polynucleotide sequence is contained within a host cell.
  • the invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment thereof, the method comprising the steps of: a) culturing the host cell containing an expression vector containing at least a fragment of the polynucleotide sequence encoding HCRP- 1 under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
  • the invention also provides a pharmaceutical composition comprising a substantially purified HCRP-1 having the amino acid sequence of SEQ ID NOJ in conjunction with a suitable pharmaceutical carrier. Still further, the invention provides a purified agonist which modulates the activity of the polypeptide of SEQ ID NO: 1.
  • the invention also provides a method for treating or preventing cancer comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising purified HCRP- 1.
  • the invention also provides a method for treating or preventing an immune disorder comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising purified HCRP-1.
  • the invention also provides a method for detecting a polynucleotide which encodes HCRP-1 in a biological sample comprising the steps of: a) hybridizing a polynucleotide sequence complementary to HCRP-1 (SEQ ID NOJ) to nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding
  • the nucleic acid material of the biological sample is amplified by the polymerase chain reaction.
  • the invention also features a substantially purified polypeptide, cyclin related protein
  • HCRP-2 having the amino acid sequence shown in SEQ ID NOJ, or fragments thereof.
  • the invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or fragments thereof and a composition comprising said polynucleotide sequence.
  • the invention also provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the amino acid sequence SEQ ID NO:3, or fragments of said polynucleotide sequence.
  • the invention further provides a polynucleotide sequence comprising the complement of the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:3, or fragments or variants of said polynucleotide sequence.
  • the invention also provides an isolated and purified sequence comprising SEQ ID NOJ or variants thereof.
  • the invention provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:4.
  • the invention provides a composition comprising an isolated and purified polynucleotide sequence comprising the complement of SEQ ID NO:4, or fragments or variants thereof.
  • the invention also provides a polynucleotide sequence comprising the complement of
  • the present invention further provides an expression vector containing at least a fragment
  • the expression vector containing the polynucleotide sequence is contained within a host cell.
  • the invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:3 or a fragment thereof, the method comprising the steps of: a) culturing the host cell containing an expression vector containing at least a fragment of the polynucleotide sequence encoding HCRP-2 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 HCRP-2 having the amino acid sequence of SEQ ID NO:3 in conjunction with a suitable pharmaceutical carrier.
  • the invention provides a purified agonist which modulates the activity of the polypeptide of SEQ ID NO:3.
  • the invention also provides a purified antagonist which decreases the activity of a polypeptide of SEQ ID NO: 3.
  • the invention provides a purified antibody which binds to a polypeptide comprising at least a fragment of the amino acid sequence of SEQ ID NO:3.
  • the invention also provides a method for treating or preventing a developmental disorder comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising purified HCRP-2.
  • the invention also provides a method for treating or preventing cancer comprising administering to a subject in need of such treatment an effective amount of a purified antagonist of HCRP-2.
  • the invention also provides a method for detecting a polynucleotide which encodes HCRP-2 in a biological sample comprising the steps of: a) hybridizing a polynucleotide sequence complementary to HCRP-2 (SEQ ID NO: 3) to nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding HCRP-1 in the biological sample.
  • the nucleic acid material of the biological sample is amplified by the polymerase chain reaction.
  • Figures 1A, IB, and IC show the amino acid sequence (SEQ ID NOJ) and nucleic acid sequence (SEQ ID NO:2) of HCRP-1.
  • the alignment was produced using MacDNASIS PROTM ⁇ software (Hitachi Software Engineering Co. Ltd. San Bruno, CA).
  • Figures 2A, 2B, 2C, 2D, 2E, and 2F show the amino acid sequence (SEQ ID NO:3) and nucleic acid sequence (SEQ ID NO:4) of HCRP-2.
  • the alignment was produced using MacDNASIS PROTM software.
  • Figure 3 shows the amino acid sequence alignments among HCRP-1 (SEQ ID NOJ), the cyclin-specific ubiquitin carrier protein E2-C from atlantic clam (GI 1493838; SEQ ID NO:5) and the ubiquitin carrier protein E2-18KD from yeast (GI 171866; SEQ ID NO:6), produced using the multisequence alignment program of DNASTARTM software (DNASTAR Inc, Madison WI).
  • Figures 4A and 4B show the amino acid sequence alignments among HCRP-2 (SEQ ID NO:3), and cyclin B from mouse (GI 50613; SEQ ID NOJ) and human (GI 105763; SEQ ID NO:8), produced using the multisequence alignment program of DNASTARTM software.
  • Figures 5 A and 5B show the hydrophobicity plots for HCRP-1, SEQ ID NO: 1 and cyclin- specific ubiquitin carrier protein E2-C from atlantic clam (SEQ ID NO:5), respectively; the positive X axis reflects amino acid position, and the negative Y axis, hydrophobicity (MacDNASIS PRO software).
  • Figures 6A and 6B show the hydrophobicity plots for HCRP-2, SEQ ID NO: 3 and cyclin B from mouse (SEQ ED NOJ), respectively ; the positive X axis reflects amino acid position, and the negative Y axis, hydrophobicity (MacDNASIS PRO software).
  • HCRP refers to the amino acid sequences of substantially purified HCRP obtained from any species, particularly mammalian, including bovine, ovine, porcine, murine, equine, and preferably human, from any source whether natural, synthetic, semi-synthetic, or recombinant.
  • agonist refers to a molecule which, when bound to HCRP, increases or prolongs the duration of the effect of HCRP.
  • Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of HCRP.
  • alleles are an alternative form of the gene encoding HCRP. Alleles may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • altered nucleic acid sequences encoding HCRP as used herein include those with deletions, insertions, or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent HCRP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular ohgonucleotide probe of the polynucleotide encoding HCRP, and improper or unexpected hybridization to alleles, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding HCRP.
  • the encoded protein may also be "altered” and contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally
  • HCRP HC-deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophihcity, and/or the amphipathic nature of the residues as long as the biological or immunological activity of HCRP 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 hydrophihcity values may include leucine, isoleucine, and valine, glycine and alanine, asparagine and glutamine, serine and threonine, and phenylalanine and tyrosine.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragment thereof, and to naturally occurring or synthetic molecules. Fragments of HCRP are preferably about 5 to about 15 amino acids in length and retain the biological activity or the immunological activity of HCRP. Where "amino acid sequence” is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, amino acid sequence, and like terms, are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
  • PCR polymerase chain reaction
  • Antagonist refers to a molecule which, when bound to HCRP, decreases the amount or the duration of the effect of the biological or immunological activity of HCRP. Antagonists may include proteins, nucleic acids, carbohydrates, or any other molecules which decrease the effect of HCRP.
  • the term "antibody” refers to intact molecules as well as fragments thereof, such as Fa, F(ab') 2> and Fv, which are capable of binding the epitopic determinant.
  • Antibodies that bind HCRP polypeptides can be prepared using intact polypeptides or 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. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin. The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit).
  • 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 nucleotide sequences which are complementary to a specific DNA or RNA sequence.
  • antisense strand is used in reference to a nucleic acid strand that is complementary to the "sense” strand.
  • Antisense molecules include peptide nucleic acids and 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 block either transcription or translation. The designation “negative” is sometimes used in reference to the antisense strand, and “positive” is sometimes used in reference 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 HCRP, or 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” binds to the complementary sequence "T-C-A”.
  • Complementarity between two single-stranded molecules may be "partial", in which only some of the nucleic acids bind, or it may be complete when 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 hybridization between 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 PNA molecules.
  • composition comprising a given polynucleotide sequence refers broadly to any composition containing the given polynucleotide sequence.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotide sequences encoding HCRP (SEQ ID NO: 1 or SEQ ID NO:3) or fragments thereof (e.g., SEQ ID NO:2 of SEQ ID NO:4 and fragments thereof) 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., NaCl), detergents (e.g., SDS) and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
  • salts e.g., NaCl
  • detergents e.g., SDS
  • other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • Consensus refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, has been extended using XL-PCRTM (Perkin Elmer, Norwalk, CT) in the 5' and/or the 3' direction and resequenced, or has been assembled from the overlapping sequences of more than one Incyte Clone using a computer program for fragment assembly (e.g., GELVIEWTM Fragment Assembly system, GCG, Madison, WI). Some sequences have been both extended and assembled to produce the consensus sequence .
  • correlates with expression of a polynucleotide indicates that the detection of the presence of ribonucleic acid that is similar to SEQ ID NO:2 or SEQ ID NO:4 by northern analysis is indicative of the presence of mRNA encoding HCRP in a sample and thereby correlates with expression of the transcript from the polynucleotide encoding the protein.
  • a “deletion”, as used herein, refers to a change in the amino acid or nucleotide sequence and results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to the chemical modification of a nucleic acid encoding or complementary to HCRP or the encoded HCRP. Such modifications include, for example, replacement of hydrogen by an alkyl, acyl, or amino group.
  • a nucleic acid derivative encodes a polypeptide which retains the biological or immunological function of the natural molecule.
  • a derivative polypeptide is one which is modified by glycosylation, pegylation, or any similar process which retains the biological or immunological function of the polypeptide from which it was derived.
  • low stringency conditions are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction.
  • the absence of non-specific lo 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% identity). In the absence of non-specific binding, the probe will not hybridize to the second non-complementary target sequence.
  • HACs Human artificial chromosomes
  • HACs are linear microchromosomes which may contain DNA sequences of 10K to 10M in size and contain all of the elements required for stable mitotic chromosome segregation and maintenance (Harrington, JJ. et al. (1997) Nat Genet. 15:345-355).
  • humanized antibody refers to antibody molecules in which amino acids have been replaced in the non-antigen binding regions in order to more closely resemble a human antibody, while still retaining the 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 G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions.
  • the two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration.
  • a hybridization complex may be formed in solution (e.g., C 0 t or RJ analysis) or 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).
  • Microarray refers to a high-density array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support.
  • modulate refers to a change in the activity of HCRP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional or immunological properties of HCRP.
  • Nucleic acid sequence refers to an ohgonucleotide, nucleotide, or polynucleotide, and fragments thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.
  • “Fragments” are those nucleic acid sequences which are greater than 60 nucleotides than in length, and most
  • H preferably includes fragments that are at least 100 nucleotides or at least 1000 nucleotides, and at least 10.000 nucleotides in length
  • ohgonucleotide refers to a nucleic acid sequence of at least about 6 nucleotides to about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20 to 25 nucleotides, which can be used in PCR amplification or hybridization assays. As used herein, ohgonucleotide is substantially equivalent to the terms “amphmers”," primers", “oligomers”, and “probes”, as commonly defined in the art.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an ohgonucleotide of at least five nucleotides in length linked to a peptide backbone of amino acid residues which ends in lysine.
  • the terminal lysine confers solubility to the composition PNAs may be pegylated to extend their hfespan in the cell where they preferentially bind complementary single stranded DNA and RNA and stop transcript elongation (Nielsen. P.E. et al. (1993) Anticancer Drug Des. 8:53-63).
  • portion refers to fragments of that protein The fragments may range in size from five amino acid residues to the entire amino acid sequence minus one amino acid
  • a protein "comprising at least a portion of the amino acid sequence of SEQ ID NO- 1 or SEQ ID NO 3" encompasses the full-length HCRP and fragments thereof
  • sample is used in its broadest sense
  • a biological sample suspected of containing nucleic acid encoding HCRP, or fragments thereof, or HCRP itself may comprise a bodily fluid, extract from a cell, chromosome, organelle, or membrane isolated from a cell, a cell, genomic DNA, RNA, or cDNA( ⁇ n solution or bound to a solid support, a tissue, a tissue print, and the like.
  • binding refers to that interaction between a protein or peptide and an agonist, an antibody and an antagonist. The interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) of the protein recognized by the binding molecule. For example, if an antibody is specific for epitope "A", the presence of a protein containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody
  • stringent conditions refer to the conditions for hybridization as defined by the nucleic acid, salt, and temperature. These conditions are well known in the art and may be altered in order to identify or detect identical or related polynucleotide sequences.
  • Numerous equivalent conditions comprising either low or high stringency depend on factors such as the length and nature of the sequence (DNA, RNA, base composition), nature of the target (DNA, RNA, base composition), milieu (in solution or immobilized on a solid substrate), concentration of salts and other components (e.g., formamide, dextran sulfate and/or polyethylene glycol), and temperature of the reactions (within a range from about 5°C below the melting temperature of the probe to about 20°C to 25°C below the melting temperature).
  • concentration of salts and other components e.g., formamide, dextran sulfate and/or polyethylene glycol
  • temperature of the reactions within a range from about 5°C below the melting temperature of the probe to about 20°C to 25°C below the melting temperature.
  • One or more factors be may be varied to generate conditions of either low or high stringency different from, but equivalent to, the above listed conditions.
  • substantially purified refers to nucleic or amino acid sequences that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 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. It may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method 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. Such
  • transformed cells include 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. They also include cells which transiently express the inserted DNA or RNA for limited periods of time.
  • a "variant" of HCRP 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 a glycine with a tryptophan.
  • Analogous minor variations may also include amino acid deletions or insertions, or both.
  • Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, DNASTAR software.
  • the invention is based on the discovery of new human cyclin related proteins (hereinafter referred to as "HCRP”), the polynucleotides encoding HCRP, and the use of these compositions for the diagnosis, prevention, or treatment of cancer, immune disorders, and developmental disorders.
  • HCRP human cyclin related proteins
  • the invention encompasses a purified related protein (HCRP-1) comprising the amino acid sequence of SEQ ID NOJ, as shown in Figure 1.
  • HCRP-1 is 179 amino acids in length and has a potential myrsitoylation site at residue G19.
  • a potential casein kinase 2 phosphorylation site is found at T72, and a potential protein kinase C phosphorylation site is found at T95.
  • a leucine zipper motif is found beginning at LI 18 and consisting of leucine residues repeated at regular intervals at LI 18, L125, L132, and L139.
  • HCRP- 1 has chemical and structural homology with the cyclin-specific ubiquitin carrier protein E2-C from atlantic clam (GI 1493838; SEQ ID NO:5) and the ubiquitin carrier protein E2-18KD from yeast (GI 171866; SEQ ID NO:6).
  • HCRP-1 shares 61% and 53% identity with E2-C and E2-18KD, respectively.
  • the active site cysteine residue of Ub-conjugating enzymes is found in HCRP-1 at Cl 14 and is conserved in both E2-C and E2-18KD.
  • the UBC domain surrounding this active site extends from approximately Y 103 to LI 18 and is also highly conserved in the other E2s.
  • HCRP-1 A proline rich region is found in HCRP-1 on the N-terminal side of the active cysteine residue. Proline residues located at P86, P90, P94, P101, and P105 in HCRP- 1 are conserved in the other E2s.
  • HCRP-1 contains a unique N-terminal extension characteristic of class IH E2s which spans residues A14 through A27. As illustrated by Figs. 5A and 5B, HCRP-1 and E2-C have rather similar hydrophobicity plots.
  • Northern analysis shows the expression of this sequence in various libraries, at least 51% of which are immortalized or cancerous and at least 24% of which involve inflammation and the immune response. Of particular note is the expression of HCRP- 1 in carcinomas of the bladder and pancreas, in metastatic tumors in the brain and lung, and in inflammed tissues associated with rehumatoid arthritis.
  • Nucleic acids encoding the HCRP- 1 of the present invention were first identified in Incyte Clone 1308478 from the fetal colon cDNA library (COLNFET02) using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:2 was derived from the following overlapping and/or extended nucleic acid sequences: Incyte Clones 993201/
  • the invention encompasses a purified cyclin related protein
  • HCRP-2 comprising the amino acid sequence of SEQ ID NO:3, as shown in Figure 2.
  • HCRP-2 is 398 amino acids in length and has a typical "cyclin box", signature sequence of 180 amino acids extending from M165 through S313. A putative "destruction box” sequence is found beginning at R32 (RTVLEEIGN).
  • HCRP-2 has chemical and structural homology with cyclin B from mouse (GI 50613; SEQ ID NOJ) and human (GI 105763; SEQ ID NO:8).
  • HCRP-2 shares 89% and 54% identity with mousse and human cyclin B, respectively.
  • the region of the "cyclin box” in HCRP-2 is well conserved in both mouse and human cyclin B.
  • the "destruction box" for ubiquitin-mediated degradation is also well conserved in the mouse and human cyclins. As shown in Figure 6A and 6B, HCRP-2 and the mouse cyclin B have rather similar hydrophobicity plots. Northern analysis shows the expression of this sequence in various libraries, at least 70% of which are immortalized or cancerous and 20% of which involve fetal tissues.
  • Nucleic acids encoding the HCRP-2 of the present invention were first identified in Incyte Clone 7755 from the human mast cell line cDNA library (HMC1NOT01) using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:4 was derived from the following overlapping and/or extended nucleic acid sequences: Incyte Clones 7755/ HMC1NOT01, 256850 and 484361/ HNT2RAT01, and 441496/ MPHGNOT03.
  • the invention also encompasses HCRP variants.
  • a preferred HCRP variant is one having at least 80%, and more preferably 90%, amino acid sequence identity to the HCRP amino acid sequence (SEQ ID NO: 1 or SEQ ED NO:3).
  • a most preferred HCRP variant is one having at least 95% amino acid sequence identity to SEQ ID NO: 1 or SEQ ID NO:3.
  • the invention also encompasses polynucleotides which encode HCRP. Accordingly, any nucleic acid sequence which encodes the amino acid sequence of HCRP can be used to produce recombinant molecules which express HCRP. In a particular embodiment, the invention encompasses the polynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 or SEQ ID NO:4 as shown in Figure 1 or Figure 3, respectively.
  • nucleotide sequences encoding HCRP may be produced.
  • the invention contemplates each and every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring HCRP, and all such variations are to be considered as
  • nucleotide sequences which encode HCRP and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring HCRP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding HCRP or its derivatives possessing a substantially different codon usage. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
  • 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, or fragments thereof, which encode HCRP and its derivatives, entirely by synthetic chemistry.
  • the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents that are well known in the art.
  • synthetic chemistry may be used to introduce mutations into a sequence encoding HCRP or any fragment thereof
  • polynucleotide sequences that are capable of hybridizing to the claimed nucleotide sequences, and in particular, those shown in SEQ ED NO.2 or SEQ ED NO 4, under various conditions of stringency as taught in Wahl, G.M. and S.L. Berger (1987, Methods Enzymol. 152:399-407) and Kimmel, A.R (1987. Methods Enzymol 152:507- 511)
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, Sequenase® (US Biochemical Corp, Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,
  • the process is automated with machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • nucleic acid sequences encoding HCRP may be extended utilizing a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, one method which may be
  • Ik- employed, "restriction-site" PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, G. (1993) PCR Methods Applic. 2:318-322).
  • genomic DNA is first amplified in the presence of primer to a linker sequence and a primer specific to the known region.
  • the amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one.
  • Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR may also be used to amplify or extend sequences using divergent primers based on a known region (Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186).
  • the primers may be designed using commercially available software such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison, MN), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68°-72° C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic. 1: 111-119).
  • capture PCR involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic. 1: 111-119).
  • multiple restriction enzyme digestions and ligations may also be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
  • Another method which may be used to retrieve unknown sequences is that of Parker, J.D. et al. (1991: Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PromoterFinderTM libraries to walk genomic DNA (Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions. When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Also, random-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 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 fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled devise camera.
  • 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 the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
  • polynucleotide sequences or fragments thereof which encode HCRP may be used in recombinant DNA molecules to direct expression of HCRP, fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced, and these sequences may be used to clone and express HCRP. As will be understood by those of skill in the art, it may be advantageous to produce
  • HCRP-encoding nucleotide sequences possessing non-naturally occurring codons For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
  • the nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter HCRP encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences.
  • site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • natural, modified, or recombinant nucleic acid sequences encoding HCRP may be ligated to a heterologous sequence to encode a fusion protein.
  • a heterologous sequence to encode a fusion protein.
  • a fusion protein may also be engineered to contain a cleavage site located between the HCRP encoding sequence and the heterologous protein sequence, so that HCRP may be cleaved and purified away from the heterologous moiety.
  • sequences encoding HCRP may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, M.H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).
  • the protein itself may be produced using chemical methods to synthesize the amino acid sequence of HCRP, or a fragment thereof.
  • peptide synthesis can be performed using various solid-phase techniques (Roberge, J.Y. et al. (1995) Science 269:202-204) and automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer).
  • the newly synthesized peptide may be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, T. (1983) Proteins. Structures and
  • composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra). Additionally, the amino acid sequence of HCRP, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
  • nucleotide sequences encoding HCRP or functional equivalents may be inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding HCRP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook, J. et al. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press, Plainview, NY, and Ausubel, F.M. et al. (1989) Current Protocols in Molecular Biology. John Wiley & Sons, New York, NY.
  • a variety of expression vector/host systems may be utilized to contain and express sequences encoding HCRP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; 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.
  • control elements are those non-translated regions of the vector— enhancers, promoters, 5' and 3' untranslated regions— which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the Bluescript® phagemid (Stratagene, LaJolla, CA) or pSportlTM plasmid (Gibco BRL) and the like may be used.
  • inducible promoters such as the hybrid lacZ promoter of the Bluescript® phagemid (Stratagene, LaJolla, CA) or pSportlTM plasmid (Gibco BRL) and the like may be used.
  • the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO; and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding HCRP, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
  • Promoters or enhancers derived from the genomes of plant cells e.g., heat shock, RUBISCO; and storage protein genes
  • plant viruses e.g., viral promoters or leader sequences
  • a number of expression vectors may be selected depending upon the use intended for HCRP.
  • vectors which direct high level expression of fusion proteins that are readily purified may be used.
  • Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as Bluescript® (Stratagene), in which the sequence encoding HCRP may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced; pEN vectors (Van Heeke. G. and S.M. Schuster (1989) J. Biol. Chem.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • Saccharomyces cerevisiae Saccharomyces cerevisiae.
  • the expression of sequences encoding HCRP may be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. ( 1987) EMBO J. 3:131 1).
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3: 1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ.
  • constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection.
  • pathogen-mediated transfection Such techniques are described in a number of generally available reviews (see, for example, Hobbs, S. or Murry, L.E. in McGraw Hill Yearbook of Science and Technology ( 1992) McGraw Hill, New York, NY; pp. 191 - 196.
  • An insect system may also be used to express HCRP.
  • Autographa califomica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the sequences encoding HCRP may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of HCRP will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses may then be used to infect, for example, S.
  • HCRP frugiperda cells or Trichoplusia larvae in which HCRP may be expressed
  • a number of viral-based expression systems may be utilized.
  • sequences encoding HCRP 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 a viable virus which is capable of expressing HCRP in infected host cells (Logan, J.
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • HACs Human artificial chromosomes
  • HACs may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
  • HACs of 6 to 10M are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding HCRP. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding HCRP, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20: 125-162).
  • a host cell strain may be chosen for its ability to modulate the 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 facilitate correct insertion, folding and/or function.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities 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
  • cell lines which stably express HCRP may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
  • any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) genes which can be employed in tk " or aprt " cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
  • npt which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150: 1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman, S.C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci.
  • marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed.
  • sequence encoding HCRP is inserted within a marker gene sequence
  • transformed cells containing sequences encoding HCRP can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding HCRP 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 which contain the nucleic acid sequence encoding HCRP and express HCRP may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein.
  • polynucleotide sequences encoding HCRP can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding HCRP.
  • Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequences encoding HCRP to detect transformants containing DNA or RNA encoding HCRP.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding HCRP include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide.
  • sequences encoding HCRP, 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
  • Suitable reporter molecules or labels 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 HCRP may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a transformed cell may be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode HCRP may be designed to contain signal sequences which direct secretion of HCRP through a prokaryotic or eukaryotic cell membrane.
  • Other constructions may be used to join sequences encoding HCRP to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins.
  • Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Emmunex Corp., Seattle, WA).
  • metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals
  • protein A domains that allow purification on immobilized immunoglobulin
  • the domain utilized in the FLAGS extension/affinity purification system (Emmunex Corp., Seattle, WA).
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and HCRP may be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing HCRP and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site.
  • the histidine residues facilitate purification on IMAC (immobilized metal ion affinity 1 chromatography as described in Porath, J. et al. (1992, Prot. Exp. Purif. 3: 263-281) while the enterokinase cleavage site provides a means for purifying HCRP from the fusion protein.
  • IMAC immobilized metal ion affinity 1 chromatography as described in Porath, J. et al. (1992, Prot. Exp. Purif. 3: 263-281
  • the enterokinase cleavage site provides a means for purifying HCRP from the fusion protein.
  • fragments of HCRP may be produced by direct peptide synthesis using solid-phase techniques Merrifield J. ( 1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Various fragments of HCRP may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.
  • HCRP- 1 Chemical and structural homology exits among HCRP- 1 and the cyclin-specific ubiquitin carrier protein E2-C from atlantic clam (GI 1493838) and the ubiquitin carrier protein E2-18KD from yeast (GI 171866).
  • HCRP is expressed in cancerous tissues and tissues associated with inflammation and the immune response. Therefore, HCRP-1 appears to play a role in cancer and immune disorders. In particular, a decrease in the level or activity of HCRP- 1 appears to be associated with the development of cancer or immune disorders.
  • HCRP-1 or a fragment or derivative thereof may be administered to a subject to prevent or treat cancer.
  • Cancers may include, but are not limited to, adenocarcinoma. leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma and particularly cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.
  • a vector capable of expressing HCRP- 1 , or a fragment or a derivative thereof may also be administered to a subject to prevent or treat cancer, including those described above.
  • an agonist which modulates the activity of HCRP- 1 may also be administered to a subject to prevent or treat cancer such as those described above.
  • HCRP- 1 or a fragment or derivative thereof may be administered to a subject to prevent or treat an immune disorder.
  • Such disorders may include, but are not limited to, A DS, Addison's disease, adult respiratory distress syndrome, allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitus, Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, atrophic gastritis, glomerulonephritis, gout, Graves' disease, hypereosinophilia.
  • irritable bowel syndrome lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis, scleroderma, Sj ⁇ gren's syndrome, and autoimmune thyroiditis; complications of cancer, hemodialysis, extracorporeal circulation; viral, bacterial, fungal, parasitic, protozoal, and helminthic infections and trauma.
  • a vector capable of expressing HCRP-1, or a fragment or a derivative thereof may also be administered to a subject to prevent or treat an immune disorder, including those described above.
  • an agonist which modulates the activity of HCRP-1 may also be administered to a subject to prevent or treat an immune disorder such as those described above.
  • HCRP-2 Chemical and structural homology exits among HCRP-2 and cyclin B from mouse (GI 506137) and human (GI 105763).
  • HCRP-2 is expressed in cancerous tissues and fetal tissues. Therefore, HCRP-2 appears to play a role in cancer and developmental disorders.
  • an increase in the level or activity of HCRP-2 appears to be associated with cancer, while a decrease in the level or activity of HCRP-2 appears to be associated with developmental disorders. Therefore, in another embodiment, HCRP-2 or a fragment or derivative thereof may be administered to a subject to prevent or treat a developmental disorder.
  • developmental disorder refers to any disorder associated with development of an organ or organ system of a subject, i.e., adrenal gland, skeletal system, reproductive system, etc.
  • Such disorders include, but are not limited to, renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, epilepsy, gonadal dysgenesis, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spinal bifida, and congenital glaucoma, cataract, or sensorineural hearing loss.
  • a vector capable of expressing HCRP-2, or a fragment or a derivative thereof may also be administered to a subject to prevent or treat a developmental disorder, including those described above.
  • an agonist which modulates the activity of HCRP-2 may also be administered to a subject to prevent or treat a developmental disorder, including those described above.
  • antagonists which decrease the activity of HCRP-2 may be administered to a subject to prevent or treat cancer.
  • Cancers may include, but are not limited to, adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma and particularly cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.
  • antibodies which specifically bind HCRP-2 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 HCRP-2.
  • a vector expressing the complement of the polynucleotide encoding HCRP-2 may be administered to a subject to treat or prevent cancer, including those 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.
  • Antagonists or inhibitors of HCRP may be produced using methods which are generally known in the art.
  • purified HCRP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind HCRP.
  • Antibodies to HCRP may be generated using methods that are well known in the art.
  • Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, 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 HCRP or 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, pluromc polyols, polyamons, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette-Gue ⁇ n
  • Corynebacte ⁇ um parvum are especially preferable.
  • the ohgopeptides, peptides, or fragments used to induce antibodies to HCRP have an amino acid sequence consisting of at least five amino acids and more preferably at least 10 ammo acids. It is also preferable that they are identical to a portion of the amino acid sequence of the natural protein, and they may contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of HCRP amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chime ⁇ c molecule.
  • Monoclonal antibodies to HCRP 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- hyb ⁇ doma technique (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; Cole, S.P. et al. (1984) Mol. Cell Biol. 62: 109-120).
  • chimeric antibodies the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison, S.L. et al. (1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).
  • techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce HCRP-specific single chain antibodies.
  • Antibodies with related specificity, but of distinct ldiotypic composition may be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton D.R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).
  • 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 (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G. et al. (1991) Nature 349:293-299).
  • Antibody fragments which contain specific binding sites for HCRP may also be generated.
  • fragments include, but are not limited to, the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be 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 (Huse, W.D. et al. ( 1989) Science 254: 1275-1281).
  • Various immunoassays may be used for screening to identify antibodies having the desired specificity.
  • Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.
  • Such immunoassays typically involve the measurement of complex formation between HCRP and its specific antibody.
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering HCRP epitopes is preferred, but a competitive binding assay may also be employed (Maddox, supra).
  • the polynucleotides encoding HCRP, or any fragment or complement thereof may be used for therapeutic purposes.
  • the complement of the polynucleotide encoding HCRP 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 HCRP.
  • complementary molecules or fragments may be used to modulate HCRP 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 HCRP.
  • Expression vectors derived from retro viruses, adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population. Methods which are well known to those skilled in the art can be used to construct vectors which will express nucleic acid sequence which is complementary to the polynucleotides of the gene encoding HCRP. These techniques are described both in Sambrook et al. (supra) and in Ausubel et al. (supra). Genes encoding HCRP can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide or fragment thereof which encodes HCRP.
  • 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 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 HCRP (signal sequence, promoters, enhancers, and introns). Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J.E. et al.
  • the 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. Examples which may be used include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding HCRP.
  • Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC.
  • 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 ohgonucleotide 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 HCRP. 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
  • W linkages within the backbone of the molecule This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine. queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
  • nontraditional bases such as inosine. queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
  • 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 polycationic amino polymers (Goldman, C.K. et al. (1997) Nature Biotechnology 15:462-66; incorporated herein by reference) may be achieved using methods which are well known in the art.
  • compositions may consist of HCRP, antibodies to HCRP, mimetics, agonists, antagonists, or inhibitors of HCRP.
  • the compositions may be administered alone or in combination with at least one other agent, such as 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.
  • 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 combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from com, 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, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler 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 or triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers may also be used for delivery.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to 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 lyophihzing 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, succinic, etc. 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: 1-50 mM histidine, 0J%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • compositions After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition.
  • labeling would include amount, frequency, and method of administration.
  • 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, usually mice, rabbits, dogs, or pigs.
  • the 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 HCRP or fragments thereof, antibodies of HCRP, agonists, antagonists or inhibitors of HCRP, which ameliorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration. The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect.
  • Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
  • Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from 0J to 100,000 micrograms, up to a total dose of about 1 g, 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 HCRP may be used for the diagnosis of conditions or diseases characterized by expression of HCRP, or in assays to monitor patients being treated with HCRP, agonists, antagonists or inhibitors.
  • the antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for HCRP include methods which utilize the antibody and a label to detect HCRP in human body fluids or extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by joining them, either covalently or non- covalently, with a reporter molecule.
  • a wide variety of reporter molecules which are known in the art may be used, several of which are described above.
  • HCRP HCRP-specific reactivated protein kinase kinase
  • HCRP HCRP-specific reactivated protein kinase
  • normal or standard values for HCRP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to HCRP under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, but preferably by photometric, means. Quantities of HCRP 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 HCRP may be used for diagnostic purposes.
  • the polynucleotides which may be used include ohgonucleotide 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 HCRP may be correlated with disease.
  • the diagnostic assay may be used to distinguish between absence, presence, and excess expression of HCRP, and to monitor regulation of HCRP levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding HCRP or closely related molecules, may be used to identify nucleic acid sequences which encode HCRP.
  • the specificity of the probe whether it is made from a highly specific region, e.g., 10 unique nucleotides in the 5' regulatory region, or a less specific region, e.g., especially in the 3' coding region, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low) will determine whether the probe identifies only naturally occurring sequences encoding HCRP, alleles, or related sequences.
  • Probes may also be used for the detection of related sequences, and should preferably contain at least 50% of the nucleotides from any of the HCRP encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and derived from the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4 or from genomic sequence including promoter, enhancer elements, and introns of the naturally occurring HCRP.
  • Means for producing specific hybridization probes for DNAs encoding HCRP include the cloning of nucleic acid sequences encoding HCRP or HCRP derivatives into vectors for the production of mRNA probes.
  • Such vectors are known in the art, commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, radionuclides such as 32P or 35S, or enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotide sequences encoding HCRP may be used for the diagnosis of conditions, disorders, or diseases which are associated with expression of HCRP.
  • conditions or diseases include cancer, such as cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; immune disorders such as AIDS, Addison's disease, adult respiratory distress syndrome, allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitus, Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis, diabetes mellitus.
  • cancer such as cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, gangli
  • emphysema atrophic gastritis, glomerulonephritis, gout, Graves' disease, hypereosinophilia, irritable bowel syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis.
  • myocardial or pericardial inflammation myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, and autoimmune thyroiditis; complications of cancer, hemodialysis, extracorporeal circulation; viral, bacterial, fungal, parasitic, protozoal, and helminthic infections and trauma; and developmental disorders such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, epilepsy, gonadal dysgenesis, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spinal bifida, and congenital glaucoma, cataract, or sensorineural hearing loss.
  • the polynucleotide sequences encoding HCRP may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; or in dipstick, pin, ELISA assays or microarrays utilizing fluids or tissues from patient biopsies to detect altered HCRP expression. Such qualitative or quantitative methods are well known in the art.
  • the nucleotide sequences encoding HCRP may be useful in assays that detect activation or induction of various cancers, particularly those mentioned above.
  • the nucleotide sequences encoding HCRP 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.
  • nucleotide sequences have hybridized with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding HCRP in the sample indicates the presence of the associated disease.
  • assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring 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, which encodes HCRP, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with those from an experiment where a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for disease. Deviation between standard and subject values is used to establish the presence of disease.
  • hybridization assays may be repeated on a regular basis to evaluate whether the level of expression in the patient begins to approximate that which is observed in the normal patient.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
  • a more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
  • oligonucleotides designed from the sequences encoding HCRP may involve the use of PCR.
  • Such oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably consist of two nucleotide sequences, one with sense orientation (5'->3') and another with antisense (3' ⁇ -5J, employed under optimized conditions for identification of a specific gene or condition. The same two oligomers, nested sets of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantitation of closely related DNA or RNA sequences.
  • Methods which may also be used to quantitate the expression of HCRP include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated (Melby, P.C. et al. (1993) J.
  • oligonucleotides derived from any of the polynucleotide sequences described herein may be used as probes in microarrays.
  • the microarrays can be used to monitor the expression level of large numbers of genes simultaneously (to produce a transcript image), and to identify genetic variants, mutations and polymorphisms. This information will be useful in determining gene function, understanding the genetic basis of disease, diagnosing disease, and in developing and monitoring the activity of therapeutic agents.
  • the microarray is prepared and used according to the methods described in PCT application WO95/1 1995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated herein in their entirety by reference.
  • the microarray is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs fixed to a solid support.
  • Microarrays may contain oligonucleotides which cover the known 5', or 3', sequence, or contain sequential oligonucleotides which cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence.
  • Polynucleotides used in the microarray may be oligonucleotides that are specific to a gene or genes of interest in which at least a fragment of the sequence is known or that are specific to one or more unidentified cDNAs which are common to a particular cell type, developmental or disease state.
  • the gene of interest is examined using a computer algorithm which starts at the 5 ' or more preferably at the 3' end of the nucleotide sequence.
  • the algorithm identifies oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization.
  • the oligomers are synthesized at designated areas on a substrate using a light-directed chemical process.
  • the substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.
  • the oligomers may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference.
  • a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
  • An array may be produced by hand or using available devises (slot blot or dot blot apparatus) materials and machines (including robotic instruments) and contain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots, or any other multiple which lends itself to the efficient use of commercially available instrumentation.
  • RNA or DNA from a biological sample is made into hybridization probes.
  • the mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA).
  • aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray so that the probe sequences hybridize to complementary oligonucleotides of the microarray. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence.
  • the scanned images are examined to determine degree of complementarity and the relative abundance of each ohgonucleotide sequence on the microarray.
  • the biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations.
  • a detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large scale correlation studies on the sequences, mutations, variants, or polymorphisms among samples.
  • the nucleic acid sequences which encode HCRP may also be used to generate hybridization probes which are useful for 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, such as human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions or single chromosome cDNA libraries as reviewed in Price, CM. (1993) Blood Rev. 7:127-134, and Trask, B.J. (1991) Trends Genet.
  • Fluorescent in situ hybridization FISH as described in Verma et al. (1988) Human
  • Chromosomes A Manual of Basic Techniques. Pergamon Press, New York, NY.
  • nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier, or affected individuals.
  • In situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms, or parts thereof, by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, for example, AT to 1 lq22-23 (Gatti, R.A. et al.
  • HCRP 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 HCRP and the agent being tested, may be measured.
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO84/03564.
  • En this method as applied to HCRP large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with HCRP, or fragments thereof, and washed. Bound HCRP is then detected by methods well known in the art. Purified HCRP can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • the nucleotide sequences which encode HCRP 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.
  • COLNFET02 The COLNE ⁇ T02 cDNA library was constructed from colon tissue obtained from a 20- week-old Caucasian female fetus. The pregnant mother was treated with erythromycin for seven days in the first trimester for bronchitis (specimen #RU95- 10-0739; DAM, Exton, PA).
  • the frozen tissue was homogenized and lysed using a Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury, NJ) in guanidinium isothiocyanate solution.
  • the lysate was centrifuged over a 5.7 M CsCl cushion using an Beckman SW28 rotor in a Beckman L8-70M Ultracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm at ambient temperature.
  • the RNA was extracted with acid phenol pH 4.7, precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended in RNAse-free water, and DNase treated at 37 °C. RNA extraction and precipitation were repeated as before.
  • the mRNA was then isolated using the Qiagen Oligotex kit (QIAGEN, Inc., Chatsworth, CA) and used to construct the cDNA library.
  • mRNA was handled according to the recommended protocols in the Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Cat. #18248-013, Gibco/BRL, Gaithersburg, MD). cDNAs were fractionated on a Sepharose CE-4B column (Cat. #275105-01, Pharmacia), and those cDNAs exceeding 400 bp were ligated into pSport I. The plasmid pSport 1 was subsequently transformed into DH5aTM competent cells (Cat. #18258-012, Gibco/BRL). HMCINOT01
  • the human mast cell HMCESfOTOl cDNA library was custom constructed by Stratagene (Stratagene, La Jolla, CA 92037) using mRNA purified from cultured HMC-1 cells.
  • the library was prepared by Stratagene essentially as described.
  • the human mast cell (HMC-1) cDNA library was prepared by purifying poly(A+)RNA (mRNA) from human mast cells and then enzymatically synthesizing double stranded complementary DNA (cDNA) copies of the mRNA. Synthetic adaptor oligonucleotides were ligated onto the ends of the cDNA enabling its insertion into the lambda vector.
  • the HMC- 1 library was constructed using the Uni-ZAPTM vector system (Stratagene).
  • the HMC-1 cDNA library can be screened with either DNA probes or antibody probes and the pBluescript® phagemid (Stratagene) can be rapidly excised in vivo.
  • the custom-constructed library phage particles were infected into E. coli host strain XLl-Blue® (Stratagene).
  • Alternative unidirectional vectors might include, but are not limited to, pcDNAI (Invitrogen, San Diego, CA) and pSHlox-1 (Novagen, Madison WI).
  • the phagemid forms of individual cDNA clones were obtained by the in vivo excision process, in which the host bacterial strain was coinfected with both the lambda library phage and an f 1 helper phage. Proteins derived from both the library-containing phage and the helper phage nicked the lambda DNA, initiated new DNA synthesis from defined sequences on the lambda target DNA and created a smaller, single stranded circular phagemid DNA molecule that included all DNA sequences of the pBluescript® plasmid and the cDNA insert.
  • the phagemid DNA was secreted from the cells and purified, then used to re-infect fresh host cells, where the double stranded phagemid DNA was produced. Because the phagemid carries the gene for ⁇ -lactamase, the newly-transformed bacteria are selected on medium containing ampicillin.
  • Phagemid DNA was purified using the Magic MiniprepsTM DNA Purification System (catalogue #A7100. Promega Corp., Madison, WI 53711). The DNA was eluted from the purification resin already prepared for DNA sequencing and other analytical manipulations.
  • Phagemid DNA may also be purified using the QIAWELL-8 Plasmid or QIAGEN® DNA Purification System (QIAGEN Inc, Chatsworth, CA). The DNA was eluted from the purification resin and prepared for DNA sequencing and other analytical manipulations.
  • An alternative method for purifying phagemid DNA utilizes the Miniprep Kit available from Advanced Genetic Technologies Corp. (Gaithersburg, Maryland, Catalog No. 77468). This kit is in the 96-well format and provides enough reagents for 960 purifications. Each kit contains a recommended protocol, which is employed except for the following changes.
  • the 96 f3- wells are each filled with only 1 ml of sterile Terrific Broth (catalog #22711) with carbenicilhn at 25 mg/L and glycerol at 0.4%.
  • the bacteria are cultured for 24 hours and lysed with 60 ⁇ l of lysis buffer.
  • a centrifugation step (2900 rpm for 5 minutes) is performed before the contents of the block is added to the primary filter plate.
  • the optional step of adding isopropanol to TR1S buffer is not performed.
  • samples are transferred to a Beckman 96-well block for storage.
  • Another altrenative method for purifying phagemid DNA uses the REAL Prep 96 Plasmid Kit (Catalog #26173, QIAGEN, Inc.). This kit enables the simultaneous purification of 96 samples in a 96-well block using multi-channel reagent dispensers. The recommended protocol is employed except for the following changes: 1) the bacteria are cultured in 1 ml of sterile
  • the cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol. Biol. 94:441f), using a Hamilton Micro Lab 2200 (Hamilton, Reno, NV) in combination with Peltier Thermal Cyclers (PTC200 from MJ Research, Watertown, MA) and Applied Biosystems 377 DNA Sequencing Systems; and the reading frame was determined.
  • nucleotide sequences of the Sequence Listing or amino acid sequences deduced from them were used as query sequences against databases such as GenBank, SwissProt, BLOCKS, and Pima IE. These databases which contain previously identified and annotated sequences were searched for regions of homology (similarity) using BLAST, which stands for Basic Local Alignment Search Tool (Altschul, S.F. (1993) J. Mol. Evol. 36:290-300; Altschul et al. (1990) J. Mol. Biol. 215:403-410).
  • BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying homologs which may be of prokaryotic (bacterial) or eukaryotic (animal, fungal or plant) origin. Other algorithms such as the one described in Smith RF and TF Smith (1992; Protein Engineering 5:35-51), incorporated herein by reference, can be used when dealing with primary sequence patterns and secondary structure gap penalties. As disclosed in this application, the sequences have lengths of at least 49 nucleotides. and no more than 12% uncalled bases (where N is recorded rather than A, C, G, or T).
  • threshold was set at 10 "25 for nucleotides and 10 "14 for peptides.
  • Incyte nucleotide sequences were searched against the GenBank databases for primate (pri), rodent (rod), and mammalian sequences (mam), and deduced amino acid sequences from the same clones are searched against GenBank functional protein databases, mammalian (mamp), vertebrate (vrtp) and eukaryote (eukp), for homology.
  • HMCINOT01 Each cDNA was compared to sequences in GenBank using a search algorithm developed by Applied Biosystems and incorporated into the INHERITTM 670 sequence analysis system.
  • Pattern Specification Language (TRW Enc, Los Angeles, CA) was used to determine regions of homology.
  • the three parameters that determine how the sequence comparisons run were window size, window offset, and error tolerance.
  • the DNA database was searched for sequences containing regions of homology to the query sequence, and the appropriate sequences were scored with an initial value. Subsequently, these homologous regions were examined using dot matrix homology plots to distinguish regions of homology from chance matches. Smith-Waterman alignments were used to display the results of the homology search.
  • Peptide and protein sequence homologies were ascertained using the INHERIT- 670 sequence analysis system using the methods similar to those used in DNA sequence homologies.
  • Pattern Specification Language and parameter windows were used to search protein databases for sequences containing regions of homology which were scored with an initial value. Dot-matrix homology plots were examined to distinguish regions of significant homology from chance matches.
  • BLAST which stands for Basic Local Alignment Search Tool (Altschul, S.F. (1993) J. Mol. Evol. 36:290-300; Altschul et al. (1990) J. Mol. Biol. 215:403-410), was used to search for local sequence alignments.
  • BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying homologs. BLAST is useful for matches which do not contain gaps.
  • the fundamental unit of BLAST algorithm output is the High-scoring Segment Pair (HSP).
  • An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user.
  • the BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance.
  • the parameter E establishes the statistically significant threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output.
  • 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 (Sambrook et al., supra).
  • BLAST Altschul, S.F. 1993 and 1990, supra
  • BLAST Altschul, S.F. 1993 and 1990, supra
  • This analysis is much faster than multiple, membrane-based hybridizations.
  • the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or homologous.
  • the basis of the search is the product score which is defined as: % sequence identity x % maximum BLAST score
  • 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 1-2% error; and at 70, the match will be exact. Homologous molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.
  • the nucleic acid sequence of the Incyte Clones 7755 and 1308478 were used to design ohgonucleotide primers for extending a partial nucleotide sequence to full length.
  • One primer was synthesized to initiate extension in the antisense direction, and the other was synthesized to extend sequence in the sense direction.
  • Primers were used to facilitate the extension of the known sequence "outward" generating amplicons containing new, unknown nucleotide sequence for the region of interest.
  • the initial primers were designed from the cDNA using OLIGO 4.06 (National Biosciences), or another appropriate program, to be about 22 to about 30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures of about 68° to about 72° C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
  • PCR High fidelity amplification was obtained by following the instructions for the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme and reaction mix. Beginning with 40 pmol of each primer and the recommended concentrations of all other components of the kit, PCR was performed using the Peltier Thermal Cycler (PTC200; MJ. Research, Watertown, MA) and the following parameters:
  • Step 1 94° C for 1 min (initial denaturation)
  • Step 2 65 ° C for 1 min
  • Step 3 68° C for 6 min
  • Step 4 94° C for 15 sec
  • Step 6 68° C for 7 min
  • Step 7 Repeat step 4-6 for 15 additional cycles
  • Step 8 94° C for 15 sec
  • Step 10 68° C for 7: 15 min
  • Step 11 Repeat step 8-10 for 12 cycles Step 12 72° C for 8 min
  • Step 13 4° C (and holding) A 5-10 ⁇ l aliquot of the reaction mixture was analyzed by electrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gel to determine which reactions were successful in extending the sequence. Bands thought to contain the largest products were excised from the gel, purified using QIAQuickTM (QIAGEN Inc., Chatsworth, CA), and trimmed of overhangs using Klenow enzyme to facilitate religation and cloning.
  • QIAQuickTM QIAGEN Inc., Chatsworth, CA
  • 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 4 72° C for 90 sec Step 5 Repeat steps 2-4 for an additional 29 cycles
  • nucleotide sequence of SEQ ID NO:2 or SEQ ED NO:4 is used 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:2 Oor SEQ ID NO:4 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base-pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmol of each oligomer and 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham) and T4 polynucleotide kinase (DuPont NEN ® , Boston, MA).
  • the labeled oligonucleotides are substantially purified with Sephadex G- 25 superfine resin column (Pharmacia & Upjohn). A 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 ⁇ , Eco RI, Pst I, Xba 1, or Pvu H; DuPont NEN ® ).
  • the DNA from each digest is fractionated on a 0.7 percent agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham, NH). Hybridization is carried out for 16 hours at 40°C To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0J x saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT ARTM film (Kodak, Rochester, NY) is exposed to the blots in a Phosphoimager cassette (Molecular Dynamics, Sunnyvale, CA) for several hours, hybridization patterns are compared visually.
  • oligonucleotides for a microarray the nucleotide sequence described herein is examined using a computer algorithm which starts at the 3' end of the nucleotide sequence.
  • the algorithm identifies oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that would interfere with hybridization.
  • the algorithm identifies 20 sequence-specific oligonucleotides of 20 nucleotides in length (20-mers). A matched set of oligonucleotides is created in which one nucleotide in the center of each sequence is altered.
  • a chemical coupling procedure and an ink jet device are used to synthesize oligomers on the surface of a substrate (Baldeschweiler, J.D. et al., PCT/WO95/25116, incorporated herein by reference).
  • a "gridded" array analogous to a dot (or slot) blot is used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
  • An array may be produced by hand or using available materials and machines and contain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots.
  • the microarray is washed to remove nonhybridized probes, and a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each ohgonucleotide sequence on the micro-array.
  • Sequence complementary to the HCRP-encoding sequence, or any part thereof, is used to decrease or inhibit expression of naturally occurring HCRP.
  • oligonucleotides comprising from about 15 to about 30 base-pairs is described, essentially the same procedure is used with smaller or larger sequence fragments.
  • Appropriate oligonucleotides are designed using Oligo 4.06 software and the coding sequence of HCRP, SEQ ID NO: 1.
  • a complementary ohgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
  • a complementary ohgonucleotide is designed to prevent ribosomal binding to the HCRP-encoding transcript.
  • HCRP HCRP-derived neuropeptide derived from E. coh.
  • this vector contains a promoter for ⁇ -galactosidase, followed by sequence containing the amino-terminal Met, and the subsequent seven residues of ⁇ -galactosidase.
  • a bacteriophage promoter useful for transcription and a linker containing a number of unique restriction sites.
  • Enduction of an isolated, transformed bacterial strain with EPTG using standard methods produces a fusion protein which consists of the first eight residues of ⁇ -galactosidase, about 5 to 15 residues of linker, and the full length protein.
  • the signal residues direct the secretion of HCRP into the bacterial growth media which can be used directly in the following assay for activity.
  • HCRP-1 activity is measured by the cyclin-ubiquitin hgation assay (Aristarkhov et al., supra).
  • the reaction contains in a volume of 10 ⁇ l, 40 mM Tris.HCl (pH 7.6), 5 mM Mg Cl 2 , 0.5 mM ATP, 10 mM phosphocreatine, 50 ⁇ g of creadne phosphokinase/ ml, 1 mg reduced carboxymethylated bovine serum albumin/ ml, 50 ⁇ M ubiquitin, 1 ⁇ M ubiquitin aldehyde, 1-2 pmol 125 I-labeled cyclin B, 1 pmol El, 1 ⁇ M okadaic acid, 10 ⁇ g of protein of M-phase fraction 1A (containing active E3-C and essentially free of E2-C), and varying amounts of HCRP-1.
  • the reaction is incubated at 18 °C for 60 minutes. Samples are then separated by electrophoresis on SDS/12% polyacrylamide gel. The amount of 125 I- cyclin-ubiquitin formed is quantified by Phosphorlmager analysis. The amount of cyclin-ubiquitin formation is proportional to the amount of HCRP- 1 in the reaction. HCRP-2
  • HCRP-2 activity is measured in the same assay described above, in which variable amounts of 125 I-labeled HCRP-2 are substituted for the fixed amount of 125 I-labeled cyclin B used, and a fixed amount of purified E2-C is used in place of variable amounts of HCRP-1. Under these conditions, the amount of ubiquitin-HCRP-2 formation is proportional to the amount of HCRP-2 in the reaction.
  • HCRP that is substantially purified using PAGE electrophoresis (Sambrook, supra), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
  • the amino acid sequence deduced from SEQ ID NO:2 is analyzed using DNASTAR 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. Selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions, is described by Ausubel et al. (supra), and others.
  • the oligopeptides are 15 residues in length, synthesized using an Applied Biosystems Peptide Synthesizer Model 431 A using fmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma, St. Louis, MO) by reaction with N-maleimidobenzoyl-N- hydroxysuccinimide ester (MBS; Ausubel et al., supra). Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant.
  • KLH keyhole limpet hemocyanin
  • MBS N-maleimidobenzoyl-N- hydroxysuccinimide ester
  • the resulting antisera are tested for antipepdde activity, for example, by binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio iodinated, goat anti-rabbit IgG.
  • Naturally occurring or recombinant HCRP is substantially purified by immunoaffinity chromatography using antibodies specific for HCRP.
  • An immunoaffinity column is constructed by covalently coupling HCRP 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.
  • HCRP Media containing HCRP is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of HCRP (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/HCRP binding (eg, a buffer of pH 2-3 or a high concentration of a chaotrope, such as urea or thiocyanate ion), and HCRP is collected.
  • HCRP or biologically active fragments thereof are labeled with 125 I Bolton-Hunter reagent (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 HCRP, washed and any wells with labeled HCRP complex are assayed. Data obtained using different concentrations of HCRP are used to calculate values for the number, affinity, and association of HCRP with the candidate molecules.
  • Gly lie Ser Ala Phe Pro Glu Ser Asp Asn Leu Phe Lys Trp Val Gly
  • 100 105 110 lie Cys Leu Asp He Leu Lys Glu Lys Trp Ser Ala Leu Tyr Asp Val
  • TTGTGTCGTC TTTTTAATTT TTCCTTAGAT GGTCTGTCCT TTTTGTGATT TCTGTATAGG 720
  • ATCTATCAGT ATCTCAGGCA GCTGGAGGTT TTGCAGTCCA TAAACCCACA TTTCTTAGAT 600
  • GGAAGAGATA TAAATGGACG CATGCGTGCC ATCCTAGTGG ATTGGCTGGT ACAAGTCCAC 660
  • AAAAAGGGGG CGGCCCCTCA AAAGGATCCC CCGGGGGGCC CATTTTTTCC CTTGCAGGCA 1560

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Abstract

L'invention a trait à des protéines humaines liées aux cyclines (HCRP) et à des polynucléotides identifiant et codant pour HCRP. L'invention concerne également des vecteurs, des cellules hôtes, des agonistes, des anticorps et des antagonistes. L'invention se réfère également à des méthodes permettant de traiter des troubles associés à l'expression de HCRP.
EP98930079A 1997-06-05 1998-06-05 Proteines liees aux cyclines Withdrawn EP1003880A2 (fr)

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