WO1996039427A1 - Ligand-stimulated gene expression - Google Patents

Ligand-stimulated gene expression Download PDF

Info

Publication number
WO1996039427A1
WO1996039427A1 PCT/US1996/009194 US9609194W WO9639427A1 WO 1996039427 A1 WO1996039427 A1 WO 1996039427A1 US 9609194 W US9609194 W US 9609194W WO 9639427 A1 WO9639427 A1 WO 9639427A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
polypeptide
leu
protein
amino acid
Prior art date
Application number
PCT/US1996/009194
Other languages
French (fr)
Inventor
Kendall A. Smith
Carol Beadling
Original Assignee
Trustees Of Dartmouth College
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from US08/465,585 external-priority patent/US6027914A/en
Priority claimed from US08/461,379 external-priority patent/US5871961A/en
Priority claimed from US08/462,390 external-priority patent/US5882894A/en
Priority claimed from US08/462,337 external-priority patent/US6051398A/en
Priority claimed from US08/463,081 external-priority patent/US5871960A/en
Priority claimed from US08/463,074 external-priority patent/US6020155A/en
Application filed by Trustees Of Dartmouth College filed Critical Trustees Of Dartmouth College
Priority to EP96921319A priority Critical patent/EP0832117A1/en
Publication of WO1996039427A1 publication Critical patent/WO1996039427A1/en

Links

Classifications

    • 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
    • C07K14/4718—Cytokine-induced proteins
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Mammalian cell growth, differentiation, and migration are directed by hormones and specific protein ligands, often termed cytokines.
  • cytokines include hormones and specific protein ligands, often termed cytokines.
  • cells comprising the neuroendocrine, hematopoietic and the immune/inflammatory systems are known to be governed by cytokines.
  • Cytokines like other ligands, interact with cells by means of specific receptors, usually expressed on the cell surface.
  • a fundamental problem confronting biomedical scientists is to discern how signals are transduced through ligand receptors and how these signals determine the response of the cell.
  • Many ligands influence their target cells by stimulating the expression of specific genes.
  • the genes signaled by most cytokines remain largely unknown owing to the complexity of cellular biochemistry.
  • the gene products that are vital for performing different cellular processes are often only expressed transiently, and/or in very low concentrations so that they are difficult to detect, isolate and characterize.
  • Interleukin-2 is a cytokine that is critical for the immune system: it directs the proliferation and differentiation of T lymphocytes (T-cells), B lymphocytes (B-cells), and natural killer (NK) cells. Just how IL-2 signals these cellular events in the various types of target cells remains unknown.
  • T-cells T lymphocytes
  • B-cells B lymphocytes
  • NK natural killer cells.
  • IL-2 signals these cellular events in the various types of target cells remains unknown.
  • a few genes have been identified that are expressed as a result of IL-2 stimulation of T cells. These include the cellular protooncogenes c-fos, c-myb, c-myc, pim-1, and c-raf-1. However, exactly how many and what other genes are expressed as a result of IL- 2/IL-2 receptor interaction remains unknown.
  • This invention pertains to complementary deoxyribonucleic acid (cDNA) libraries enriched in clones containing genes induced by ligand stimulation of a cell having a corresponding receptor for the ligand, and to methods of producing the same.
  • This invention also relates to the genes which are expressed immediately or early on as a consequence of such a ligand-receptor interaction, and to methods of identifying these genes.
  • a cDNA library highly enriched in ligand-inducible genes is produced by activating a cellular receptor with a ligand to induce the expression of genes as a result of ligand-receptor binding, reverse transcribing these RNA, and differentially probing the cDNA and selecting clones that bind to induced cDNA, but not to uninduced cDNA.
  • Useful ligands include any of those which can activate a specific cellular receptor, including natural or synthetic ligands for the receptor, e.g., cytokines such as the interleukins, cellular growth factors, colony stimulating factors, hormones, peptides, antibodies, and receptor-binding fragments thereof.
  • the present invention relates to a cDNA library (ies) of nucleic acids induced by a specific ligand (s) and/or all redundant DNA sequences encoding the CR proteins, homologues and fragments, to a vector (s) carrying the library (ies), and to transfected cells carrying the hybrid vector (s) with the cloned library (ies).
  • the invention also relates to specific cDNA sequences induced by interleukin-2 (IL-2) and all other DNA sequences encoding their expressed proteins, homologues and fragments, to the corresponding RNA sequences, to the polypeptides expressed by the sequences of the cDNA library, to analogues having about 60 to 99% homology with the nucleic acid and/or the polypeptide sequences thereof, and to fragments thereof about 1 to 200 amino acids long, and in some instances even longer.
  • IL-2 interleukin-2
  • a most preferred embodiment of the present invention relates to the Cytokine Response nucleic acids CRI, CR2, CR3, CR5, CR6, CR7, and CR8 identified below as SEQ. ID No: 1, 3, 5, 7, 9, 11, and 27, and all other nucleic acids encoding the same proteins, and to the respe ⁇ ive proteins encoded by these nucleic acids identified below as SEQ. ID No: 2, 4, 6, 8, 10, 12, 14, or 28, and their glycosylated or deglycosylated analogues, homologues, and fragments.
  • This invention also encompasses all DNA and RNA probes of smaller lengths encoding portions of the polypeptides of this invention.
  • the genes and polypeptides of the invention may be utilized as diagnostic or therapeutic agents.
  • the genes are suitable as targets for the development of assays to discover new drugs.
  • the therapeutic agents are suitable for regulation of cellular proliferation and differentiation and, therefore, for the treatment of all kinds of cancers, immune diseases, such as allergic, autoimmune, and rheumatologic diseases, transplant reje ⁇ ion, and as anti-infe ⁇ ives for fighting viral, ba ⁇ erial, parasitic and fungal infe ⁇ ions, among others.
  • immune diseases such as allergic, autoimmune, and rheumatologic diseases
  • transplant reje ⁇ ion transplant reje ⁇ ion
  • anti-infe ⁇ ives for fighting viral, ba ⁇ erial, parasitic and fungal infe ⁇ ions, among others.
  • a cDNA library may be constru ⁇ ed, in accordance to this invention, which is enriched in nucleic acid sequences whose expression is induced by activation of a ligand-specific cellular receptor.
  • This enriched library facilitates the identification and characterization of ligand-a ⁇ ivated genes that are triggered immediately and/or early on after receptor activation, e.g., about 2 to 4 hours after the ligand binds to its receptor. Such genes are believed to play a role in stimulating growth phase transitions and subsequent clonal expansion of a particular cell type.
  • the cells are a ⁇ ivated by a ligand in the presence of labeled RNA precursors, which are incorporated into the new RNA synthesized by the cell in response to receptor activation.
  • Labeled precursors are used in order to distinguish newly transcribed from unlabeled, preexisting RNA.
  • Preferred labeled RNA precursors include 6-thioguanine, 4-thiouridine, and tritiated uridine, but others may also be utilized.
  • a ⁇ ivation is also carried out in the presence of a substance which enhances the level of RNA in a cell.
  • Preferred substances include protein synthesis inhibitors, such as cycloheximide and puromycin, although others may be used, such as cyclic 3',5'-adenosine monophosphate (cAMP), analogs of cAMP such as dibutyryl cAMP, and other molecules which increase the intracellular level of cAMP.
  • cAMP cyclic 3',5'-adenosine monophosphate
  • the labeled RNA is then separated from the unlabeled RNA and used to prepare cDNA.
  • the cDNA is cloned into a rector to provide a library of cDNA-containing clones. This library is then screened for clones containing ligand-inducible genes.
  • the screening step includes probing the cDNA library with a DNA probe constructed from total cellular RNA or mRNA derived from (1) a ligand-induced cell and (2) an uninduced cell.
  • the library is probed under conditions such that the probe hybridizes specifically with a complementary cDNA sequence in the library.
  • the sele ⁇ ing step includes sele ⁇ ing those clones containing sequences that hybridize only with probes constructed from ligand-induced mRNA or total RNA.
  • CR Cytokine Response
  • CR4 is identical to a gene reported as SATB-1 (Dickinson, L. A. et al. (1992) Cell 70:631-645), for Special AT-rich Binding protein 1, which binds sele ⁇ ively to the nuclear matrix/scaffold- associating region of DNA.
  • CR7 also identified using the method of the invention, is identical to the putative proto-oncogene, pirn 1 , a known IL-2-induced gene.
  • the nucleic acid sequences of these CR genes, i.e., CR genes 1-8 are set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 and 27.
  • the amino acid sequences encoded by these CR genes are set forth in SEQ ID NOs.: 2, 4, 6, 8, 10. 12. 14.
  • the method of the invention may be used to create cDNA libraries of the genes induced by a ⁇ ivation of a variety of different cellular receptors.
  • the receptors may be cytoplasmic, nuclear, or cell-surface receptors, and include receptors for cytokines, hormones, fa ⁇ ors, and peptides, among other types of receptors.
  • cytokines such as the interleukins (e.g., IL-1 and IL-2), cellular growth fa ⁇ ors (e.g., platelet-derived growth fa ⁇ or (PDGF), epidermal growth fa ⁇ or (EGF), fibroblast growth fa ⁇ or (FGF), insulin-like growth fa ⁇ or (IGF)), colony stimulating fa ⁇ ors (e.g., multiplication stimulating a ⁇ ivity), and hormones (e.g., insulin, somatomedin C, and steroid hormones) are useful as a ⁇ ivators of certain cellular receptors.
  • the ligand used to activate the receptor may be the natural ligand recognized by the receptor or a synthetic analogue or fragment.
  • an antibody specific for the receptor and capable of a ⁇ ivating the receptor may also be used.
  • the receptor is, thus, a ⁇ ivated by a ligand or other means of a ⁇ ivation for a predetermined length of time and at an effe ⁇ ive concentration.
  • This activation may be carried out in the presence of labeled RNA precursors which are incorporated into the RNA synthesized by the cell in response to receptor a ⁇ ivation.
  • the RNA transcribed is labeled so as to be distinguished from preexisting RNA which is not labeled.
  • Some labels (such as radiolabels) may be employed to monitor the newly synthesized RNA.
  • Useful radiolabeled RNA precursors for such purposes include [ ⁇ H]-uridine.
  • RNA synthesized from thiol-labeled RNA precursors specifically adheres to phenylmercury agarose (Woodford et al., Anal. Biochem. 1781: 166-172 (1988)).
  • RNA newly synthesized in response to receptor a ⁇ ivation may be separated from preexisting RNA in the cells. All RNA molecules expressed prior to ligand-a ⁇ ivation pass through the phenylmethyl mercury column, leaving only the newly synthesized, thiol- (SH-) labeled RNA attached to the agarose via a covalent bond between the mercury and sulfur.
  • RNA molecules may then be eluted from the column by reducing the Hg-S bond with an excess of 2-mercaptoethanol.
  • a substance that enhances the level of RNA may be added to the culture medium during the ligand stimulation (see, e.g., Cochran et al., Cell 33: 939-947 (1983)).
  • Useful substances include those compounds that stabilize RNA and/or that block translation, thereby blocking feedback inhibition of these genes by a later gene product. Such a ⁇ ivity may potentiate the magnitude of the RNA expressed as well as the duration of the life of the RNA.
  • RNA-ribosome complexing examples include cycloheximide (CHX), which inhibits protein synthesis at the level of RNA-ribosome complexing and may stabilize polysomal RNA, and puromycin, which inhibits translation by causing premature dissociation of the peptide- mRNA-ribosome complex.
  • CHX cycloheximide
  • puromycin which inhibits translation by causing premature dissociation of the peptide- mRNA-ribosome complex.
  • the labeled RNA transcribed consequent to receptor a ⁇ ivation in the presence of the substance which enhances RNA levels may then be purified from the cytoplasm of the cells.
  • the purification may be accomplished by extra ⁇ ing total cellular RNA from a cell homogenate or fraction thereof, isolating mRNA therefrom, for example, using a poly U or poly [dT] column, and then separating the labeled RNA from the unlabeled RNA.
  • the separation may be accomplished, for example, using the phen
  • a cDNA library may be prepared from the separated labeled RNA by standard techniques. For example, the labeled RNA may be reversed transcribed into cDNA, using oligo [dT] primers. The cDNA may then be ligated into appropriate ve ⁇ ors using established recombinant DNA techniques. A cDNA library is then constru ⁇ ed by methods well known in the art and transfected into prokaryotic or eukaryotic host cells. Prokaryotic systems most commonly utilize E. coli as host, although other ba ⁇ erial strains such as Bacillus, Pseudomonas, or other Gram-positive or Gram-negative prokaryotes may also be used.
  • operable control systems compatible with these hosts are ligated to the cDNA fragments and disposed on a suitable transfer ve ⁇ or which is capable of replication in the ba ⁇ erial host cell.
  • Backbone vectors capable of replication include phage and plasmid ve ⁇ ors. Common plasmid ve ⁇ ors include those derived from pBR322 and the pUC series.
  • One such useful vector which is commercially available is the plasmid p Bluescript ⁇ ISK+ (Stratagene, La Jolla, CA). Charon lambda phage is a frequently employed phage vector. Others, however, may also be employed.
  • Control sequences obligatorily include promoter and ribosome binding site encoding sequences, and a variety of such controls are available, in the an, such as the beta- la ⁇ amase (pencillinase) and la ⁇ ose (lac) promoter systems (see, e.g., Chang et al., Nature 198: 106 (1977)), and the tryptophan (trp) promoter systems (Goeddel et al., Nucleic Acids Res. 8: 4057 (1980)).
  • Tecillinase beta- la ⁇ amase
  • lac la ⁇ ose
  • trp tryptophan
  • Composite promoters containing elements of both the trp and lac promoter systems are also available in the art.
  • Eukaryotic microbes such as laboratory strains of Saccharomyces cerevisiae, or Baker's yeast, can also be used for expression.
  • yeast control systems and ve ⁇ ors are available, including those which are promoters for the synthesis of glycolytic enzymes (see, e.g., Hess et al., Biochem. 17: 4900 (1968)).
  • Yeast vectors e.g., employing the 2 micron origin of replication are suitable as transfer ve ⁇ ors (see, e.g., Broach, Meth. Enzym. 101: 307 (1982)).
  • Tissue cultures of insect cell lines, or cell lines immortalized from mammalian or other higher organisms have also been used as recombinant hosts.
  • Such cell lines include Chinese hamster ovary (CHO), Vero, HeLa, and COS cells.
  • COS cell system is used for transient expression, while CHO cells typically integrate transformed DNA into the chromosome.
  • Suitable mammalian ve ⁇ ors are generally based on viral origins of replication and control sequences. Most commonly used are the simian virus 40 (SV40) promoters and replicons (See, Fiers et al., Nature 273: 113 (1978)) and similar systems derived from Adenovirus 2, bovine papilloma virus, and avian sarcoma virus.
  • SV40 simian virus 40
  • the ligand-activated genes are then screened in the library using any one of several different methods.
  • One method involves differential hybridization with cDNA probes constructed from mRNA derived from ligand-activated cells and una ⁇ ivated cells.
  • Another method includes hybridization subtraction, whereby cDNA from ligand-a ⁇ ivated cells is hybridized with an excess of mRNA from unactivated cells to remove RNA molecules common to both.
  • cDNA probes may be made from the same pool of thiol-sele ⁇ ed mRNA used to make the cDNA library, as these sequences are highly enriched for ligand-induced molecules.
  • cDNA probes may be prepared from mRNA.
  • results from probing with the cDNA made from drug-inhibited cells may then be compared to results from probes made from cells not inhibited by these drugs.
  • drugs that block the biological response to the particular cytokine e.g., rapamycin and elevated cAMP block the proliferative response of T cells to IL-2, and cyclosporin A and FK506 block the T-cell response to activation via the T- cell antigen receptor.
  • the marked superinduction observed for a number of the genes using a substance, such as CHX, which enhances RNA levels, is crucial in enabling their detection by differential hybridization, as it has been estimated that differential hybridization is only effe ⁇ ive in the detection of relatively high-abundance RNAs expressed at a level of greater than about 500 copies per cell. Therefore, the superinduction increases that level of expression of low-abundance RNAs above the threshold of detection by differential screening. In addition, the about 10-fold enrichment for newly synthesized RNA afforded by the thiol-labeling procedure further heightens the efficacy of the cloning procedure.
  • RNA significantly enhances the sensitivity of differential screening, and provides a cloning strategy which has the capacity to dete ⁇ messages normally present in relatively low abundance (i.e., less than about 100 copies/cell).
  • all clones isolated as tentatively positive must be corroborated as truly ligand-a ⁇ ivated. This can be accomplished by isolating the cDNA insert from each cloned plasmid, and then employing this cDNA to probe RNA from ligand-a ⁇ ivated cells by Northern blot analysis. Then, to identify each gene, the cDNA may be subjected to sequence analysis.
  • GenBank Los Alamos, NM
  • EMBL Heidleberg, Germany
  • a number of methods may be used to chara ⁇ erize the novel ligand-enhanced genes and begin to determine their functional roles in, for example, signal transdu ⁇ ion.
  • An analysis of the cDNA corresponding to the mRNA transcript can be used to predi ⁇ the coding region for the gene product and its amino acid sequence. From the amino acid sequence, the gene produ ⁇ can be placed into one of several categories of proteins, such as DNA-binding proteins, kinases, phosphatases, transmembrane proteins, or secreted products.
  • the analysis of the regulatory elements governing expression of the immediate-early genes described in the present study is useful in the chara ⁇ erization of the secondary biochemical messengers activated by the ligand, e.g., IL-2 receptor.
  • Other methods helpful in determining the functional relevance of the IL-2- induced genes include examining T-cells for their expression in response to triggering of other receptors.
  • T-cell antigen receptor One such receptor is the T-cell antigen receptor.
  • Seminal studies of the T-cell system have demonstrated that T-cell a ⁇ ivation occurs as a two-step process. Quiescent cells are initially stimulated through engagement of the antigen receptor, which provides the cells with the capacity to produce and respond to IL-2. Subsequently, the interaction of IL-2 with its cell-surface receptor drives progression through the G j to the S phase of the cell cycle. Transmembrane signaling through both the T-cell antigen receptor has been shown to trigger the heightened expression of a number of genes, including c-fos, c-myc and c-raf-1 (Reed et al., P.N.A.S.
  • Additional methods that can be used to categorize the genes isolated include screening for expression by proliferating versus non-proliferating cells, for tissue-specific expression, and for expression in response to different cytokines and hormones. Genes that are expressed exclusively by proliferating cells, very likely fun ⁇ ion to promote cell growth. Other genes signal differentiation and would be expected to be tissue-specific or a ⁇ ivated only by a restri ⁇ ed family of similar ligands. An additional means of establishing the mechanisms of IL-2-mediated transmembrane signaling is provided by the varied effe ⁇ s of elevated cAMP on IL-2-induced gene expression.
  • cAMP a ⁇ ion very likely involves regulation of protein phosphorylation, as cAMP is an activator of protein kinase A, and elevations of intracellular cAMP inhibit IL-2-inducted phosphorylation events.
  • cAMP sensitivity is a useful tool with which to dissect IL-2-mediated signal transdu ⁇ ion pathways involved in cell cycle progression.
  • IL-2 induced genes encode kinases (c- raf-1, pim-1) and DNA binding proteins (c-fos, c-myc, c-myb). Sequence analysis of the novel genes then determines whether the proteins they encode contain conserved domains which would implicate similar fun ⁇ ions.
  • IL-2 stimulates cellular differentiation as well as division, and has been shown to induce the expression of a number of genes to perform roles other than in cell cycle progression.
  • cAMP sensitivity indire ⁇ ly establishes involvement in G ⁇ progression.
  • the IL-2-induced expression of the PCNA/cyclin and DNA topoisomerase II gene in late G is specifically inhibited by cAMP, so that cAMP sensitivity of immediate early gene expression provides a test for the genes that play a role in regulating late gene expression. If, like the previously characterized cell cycle regulatory cdc2/CDC28 and cyclin genes, the novel IL-2 induced genes are highly conserved, then it is possible to isolate yeast homologs of the clones and perform deletional analyses to further define the fun ⁇ ions of the gene produ ⁇ s.
  • the definitive assignment of a fun ⁇ ion to a given gene product depends upon a series of different approaches, including determining intracellular location, and determining the consequences of blocking the expression of the gene either by mRNA antisense methods or by homologous recombination methods. All of the methods necessary for these studies exist as prior art and therefore, given the identification of a given gene as a ⁇ ivated by a ligand such as the cytokine IL-2, the characterization of the product is attained.
  • CR4 is identical to a gene reported as SATB-1 (Dickinson, L. A. et al., Cell 70: 631-645 (1992)), for Special AT- rich Binding protein 1, which binds selectively to the nuclear matrix/scaffold-associating region of DNA
  • CR7 is identical to the putative proto-oncogene, pirn 1, a known IL-2-induced gene. Table I below provides several characteristics of the CR genes. Table I: CR Gene Characteristics
  • CR genes 1 to 8 The nucleic acid sequences of these CR genes, i.e., CR genes 1 to 8, are set forth in Tables II to VIII below, as SEQ. ID Nos: 1, 3, 5, 7, 9, 11, 13, and 27, or fragments thereof.
  • amino acid sequences encoded by these CR genes are also provided below as SEQ. ID Nos: 2, 4, 6, 8, 10, 12, 14, and 28.
  • the regulatory regions of all of the CR genes may be used to constru ⁇ assays to identify the relevant cis-a ⁇ ing DNA response elements, the trans-a ⁇ ing fa ⁇ ors responsible for transcriptional activation leading to CR gene expression, and the biochemical signaling for pathways triggered by IL-2 (the ligand) that a ⁇ ivate the transcriptional a ⁇ ivating fa ⁇ ors.
  • the regulatory regions of all of the CR genes may also be used to constru ⁇ assays useful for drug discovery.
  • the promoter regions of the CR genes which are 5' to the coding regions identified from the cDNA sequences, are used to constru ⁇ a promoter-reporter assay to analyze signal transdu ⁇ ion pathways.
  • Fragments of DNA derived from genomic DNA are engineered upstream from "reporters", such as luciferase. These promoter-reporter constru ⁇ s are transfe ⁇ ed into cells that are ligand-inducible, and then used as rapid and convenient assays for ligand- induced gene activation. Such assay is used in drug-discovery screening systems, where natural produ ⁇ s or synthetic chemicals are tested. Those agents showing high negative or positive a ⁇ ivity as regulators of the CR gene promoter-reporter assays, are suitable as therapeutic agents, and their chemical stru ⁇ ures may be modified by traditional medicinal chemistry, to provide congeners with further enhanced activities.
  • reporter such as luciferase.
  • the agents exhibiting a ⁇ ivity in the CR gene promoter-repo ⁇ er assays are then tested in vivo, in cellular proliferation/differentiation systems, and/or in animals and humans, for anti-proliferative a ⁇ ivity.
  • These assays can be used to identify novel agents or drugs that either suppress or a ⁇ ivate CR gene expression.
  • novel agents or drugs may be used as immunosuppressives, immunosiimulants, antiinfe ⁇ ives, or anti-cancer agents.
  • the immediate-early CR genes and gene produ ⁇ s may also be used to construct assays to determine the biochemical and molecular events, initiated by the ligand-receptor stimulation, that promote progression to the intermediate and late stages of the cell cycle responsible for DNA synthesis and replication. These assays are also used to identify novel agents and drugs that either suppress or promote these processes. With the capacity to generate large quantities of the CR gene products, the three-dimensional structures of the products are determined by conventional methods, such as x-ray crystallography and nuclear magnetic resonance. From this information, novel agents or drugs are identified, e.g., using computer analysis of the chemical structures, that interact with the CR gene product. These agents may be used as therapeutic agents.
  • the CRI gene includes 2406 nucleotides of the coding fragment of SEQ. ID NO: 1 encoding a 202 amino acid protein (about 22 RDA) of SEQ.
  • the nucleotide sequence of the CRI gene is about 58% homologous to the nucleotide sequence of the GOS8 gene (Siderovski, D. P., et al., DNA and Cell Biology 13: 125-147 (1994)), which was isolated from a PHA-induced T cell library. At the protein level, CRI is about 51.2% homologous to GOS8. In addition, the nucleotide sequence of the CRI gene is about 58% homologous to the nucleotide sequence of the BL34 gene (Hong, J.X. et al., J.
  • CRI is about 48.0% homologous to BL34.
  • the homology of CRI with BL-34 is of particular interest, in that BL-34 is expressed only by activated B cells, is preferentially expressed in vivo by B cells in lymph node germinal centers, and is overexpressed in B cell malignancies.
  • CRI is an intracellular protein.
  • the CRI protein includes no sequences consistent with other functional motifs or domains, such as found for DNA binding proteins, kinases, phosphatases, or linker molecules. The sequenced DNA and deduced protein sequence for CRI are provided below in Table II.
  • the present invention also pertains to a CRI polypeptide, preferably a substantially pure preparation of a CRI polypeptide, or a recombinant CRI polypeptide.
  • the CRI polypeptide comprises an amino acid sequence at least about 60 to 95%, preferably about 75 to 85% homologous to the amino acid sequence in SEQ ID NO: 2
  • the polypeptide has an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NO: 2
  • the polypeptide comprises at least about 5 to 200, preferably about 10 to 150, more preferably about 20 to 100 amino acids in length.
  • the polypeptide comprises at least about 5, preferably at least about 10, more preferably at least about 20, more preferably at least about 50, 100, or 150 contiguous amino acids from SEQ ID NO: 2.
  • a protein homologous to SEQ ID NO: 2 has a molecular weight of about 15-30 kD, preferably about 22 kilodaltons (kD).
  • a polypeptide having at least one biological activity of the CRI polypeptide may differ in amino acid sequence from the sequence in SEQ ID NO: 2, but such differences result in a modified polypeptide which fun ⁇ ions in the same or similar manner as or which has the same or similar characteristics of native CRI protein.
  • the CRI polypeptide is a recombinant fusion protein which includes a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated to the SEQ ID NO: 2, e.g., the second polypeptide portion comprises glutathione-S-transferase, a DNA binding domain, a polymerase activating domain so that, e.g., the fusion protein is fun ⁇ ional in a two-hybrid assay.
  • a second polypeptide portion e.g., a second polypeptide having an amino acid sequence unrelated to the SEQ ID NO: 2, e.g., the second polypeptide portion comprises glutathione-S-transferase, a DNA binding domain, a polymerase activating domain so that, e.g., the fusion protein is fun ⁇ ional in a two-hybrid assay.
  • an immunogen comprising a CRI polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the CRI polypeptide; e.g., a humoral response, an antibody response, a cellular response.
  • the immunogen comprises an antigenic determinant, e.g., a unique determinant, from a protein comprising SEQ ID NO:2 analogues or fragments thereof .
  • a further aspect of the present invention features a monoclonal or polyclonal antibody specifically rea ⁇ ive with an epitope of the CRI immunogen, which is prepared as is known in the art.
  • the subje ⁇ CRI nucleic acid includes a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the CRI gene sequence, e.g., to render the CRI gene sequence suitable for use as an expression ve ⁇ or.
  • a transcriptional regulatory sequence e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence
  • the nucleic acid which encodes a CRI polypeptide of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least about 12 to 300, more preferably to at least about 20 to 250, and more preferably to at least about 40 consecutive nucleotides of SEQ ID NO: 1.
  • the CRI encoding nucleic acid hybridizes to a nucleic acid probe corresponding to a subsequence encoding at least about 4 to 200, preferably at least about 10 to 150, and even more preferably at least about 20 to 150 amino acid residues between residues 1- 202 of SEQ ID NO: 2.
  • the nucleic acid sequence includes at least about 1 to 100, preferably about 2 to 250, more preferably about 3 to 20, still more preferably about 5 to 10 nucleotides from the region of SEQ ID NO: 1 which encodes amino acid residues 1-202 of SEQ ID NO: 2.
  • the encoded peptide includes at least about 1 to 30, preferably about 2 to 20, more preferably about 5 to 10 amino acid residues from amino acid residues 1-202 of SEQ ID NO: 2.
  • the CR2 gene comprises 1283 nucleotides, which is a fragment of SEQ. ID No: 3 encoding a small, intracellular protein of 60 amino acids (about 6.6 kD), of SEQ. ID No: 4.
  • the CR2 gene is the only CR gene for which there are no homologies to other known gene products. Elevated cAMP suppresses, but does not abolish CR2 gene expression.
  • the obtained DNA sequence and the deduced protein sequence for CR2 are provided below in Table III. Table III: Full DNA Sequence and Deduced Protein Sequence for CR2
  • AAAAAAAAAA AA (SEQ. ID No: 3) 1223
  • the present invention also pertains to a CR2 polypeptide, preferably a substantially pure preparation of a CR2 polypeptide, or a recombinant CR2 polypeptide.
  • the CR2 polypeptide comprises an amino acid sequence at least about 60% to 95%, preferably about 80% to 90% homologous to the amino acid sequence in SEQ ID NO:4, essentially the same as the SEQ ID NO:4, or fragments at least about 5 to 250, preferably about 10 to 100, and more preferably about 20 to 50 amino acids in length, at least about 5, preferably at least about 10, and more preferably at least about 20, 50, 100, or 150 contiguous amino acids from SEQ ID NO: 4.
  • a protein homologous to SEQ ID NO: 4 has a molecular weight in the range of about 5-15 kD, and more preferably about 6 kD.
  • a polypeptide having at least one biological activity of the CR2 polypeptide may differ in amino acid sequence from the sequence in SEQ ID NO: 4, but such differences result in a modified polypeptide which fun ⁇ ions in the same or similar manner as native CR2 protein or which has the same or similar characteristics of the native CR2 protein.
  • Such a peptide can include at least about 1 to 30, preferably about 2 to 20, and more preferably about 5 to 10 amino acid residues from residues 1-60 of SEQ ID NO: 4.
  • the CR2 polypeptide is a recombinant fusion protein which comprises a second polypeptide portion, e.g., a second polypeptide comprising an amino acid sequence unrelated to a protein represented by SEQ ID NO: 4, e.g., glutathione-S-transferase, a DNA binding domain, a polymerase a ⁇ ivating domain, so that the fusion protein is functional in a two-hybrid assay.
  • a second polypeptide portion e.g., a second polypeptide comprising an amino acid sequence unrelated to a protein represented by SEQ ID NO: 4, e.g., glutathione-S-transferase, a DNA binding domain, a polymerase a ⁇ ivating domain, so that the fusion protein is functional in a two-hybrid assay.
  • Yet another aspe ⁇ of the present invention concerns an immunogen comprising a CR2 polypeptide in an immunogenic preparation, the immunogen being capable of e
  • the immunogen comprises an antigenic determinant, e.g. a unique determinant, from the SEQ ID NO: 4.
  • an antigenic determinant e.g. a unique determinant, from the SEQ ID NO: 4.
  • a further aspect of the present invention features a monoclonal or polyclonal antibody preparation specifically reactive with an epitope of the CR2 immunogen.
  • nucleic acid comprising a nucleotide sequence which encodes a CR2 polypeptide, analogues and/or fragments thereof.
  • the subje ⁇ CR2 nucleic acid includes a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the CR2 gene sequence, e.g., to render the CR2 gene sequence suitable for use as an expression ve ⁇ or.
  • the nucleic acid which encodes a CR2 polypeptide hybridizes under stringent conditions to a nucleic acid probe corresponding to at least about 12 to 60, more preferably to at least about 20 to 50, still more preferably to at least about 40 to 45 consecutive nucleotides of SEQ ID NO: 3.
  • the CR2 encoding nucleic acid hybridizes to a nucleic acid probe corresponding to a subsequence encoding at least about 4 to 30, more preferably at least about 10 to 25 consecutive amino acid residues, and even more preferably at least about 20 amino acid residues between residues 1-60 of SEQ ID NO: 4.
  • the nucleic acid sequence includes at least about 1 to 100, preferably about 2 to 50, more preferably about 3 to 30, and still more preferably about 5 to 20 nucleotides from die region of SEQ ID NO: 3 which encodes amino acid residues 1-60 of SEQ ID NO: 4.
  • the encoded peptide includes at least about 1 to 30, preferably about 2 to 20, and more preferably about 3 to 10 amino acid residues 1-60 of SEQ ID NO: 4.
  • the CR3 gene includes 2451 nucleotides, shown as the fragment of SEQ. ID NO: 5 encoding a protein of 378 amino acids (about 41.5 kD) of SEQ. ID NO: 6.
  • the corresponding DNA and protein sequences corresponding to CR3 are shown in Table IV below.
  • the present invention further pertains to a CR3 polypeptide, preferably a substantially pure preparation of a CR3 polypeptide, or a recombinant CR3 polypeptide.
  • the CR3 polypeptide comprises an amino acid sequence at least about 60% to 95%. preferably about 80% to 90% homologous to the SEQ ID NO: 6.
  • the polypeptide comprises an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NO: 6, and comprises at least about 5 to 150, preferably about 10 to 100, and more preferably about 20 to 50 amino acids in length.
  • the polypeptide comprises at least about 5, preferably at least about 10, more preferably at least about 20, 50, still more preferably at least about 100, or 150 contiguous amino acids from SEQ ID NO: 6.
  • a protein homologous to SEQ ID NO: 6 has a molecular weight in the range of about 80-95 kD, preferably about 88 .
  • a peptide having at least one biological a ⁇ ivity of the CR3 polypeptide may differ in amino acid sequence from the sequence in SEQ ID NO: 6, but such differences result in a modified protein which functions in me same or similar manner as or which has the same or similar characteristics of native CR3 protein.
  • Such a peptide can include at least about 1, 2, 3, or 5, and preferably about 10, 20, and 30, amino acid residues from residues 1-358 of SEQ ID NO: 6.
  • the CR3 polypeptide is a recombinant fusion protein which comprises a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated to the SEQ ID NO: 6, e.g., the second polypeptide portion is glutathione-S- transferase, a DNA binding domain, a polymerase a ⁇ ivating domain, e.g., me fusion protein is fun ⁇ ional in a two-hybrid assay.
  • anod er aspe ⁇ of the present invention concerns an immunogen comprising a CR3 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for said CR3 polypeptide, e.g.
  • the immunogen comprises an antigenic determinant, e.g. a unique determinant from the SEQ ID NO: 6.
  • a further aspect of the present invention features a monoclonal or polyclonal antibody preparation specifically reactive with an epitope of the CR3 immunogen.
  • a substantially pure nucleic acid comprising a nucleotide sequence which encodes a CR3 polypeptide.
  • me subject CR3 nucleic acid includes a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the CR3 gene sequence, e.g., to render the CR3 gene sequence suitable for use as an expression vector.
  • the nucleic acid which encodes a CR3 polypeptide hybridizes under stringent conditions to a nucleic acid probe corresponding to at least about 12 to 300, more preferably to at least about 20 to 200, and still more preferably to at least about 40 to 100 consecutive nucleotides of SEQ ID NO: 5.
  • the CR3 encoding nucleic acid hybridizes to a nucleic acid probe corresponding to a subsequence encoding at least about 4, more preferably at least about 10, and even more preferably at least about 20 amino acid residues between residues 1-358 of SEQ ID NO: 6.
  • the nucleic acid sequence includes at least about 1, 2, 3 or 5, and preferably at least about 10, 20, 50, or 100 nucleotides from the region of SEQ ID NO: 5, which encodes amino acid residues 1-358 of SEQ ID NO: 6.
  • the encoded peptide includes at least about 1, 2, 3, 5, 10, 20, or 30 amino acid residues from amino acid residues 1-358 of SEQ ID NO: 6.
  • This protein has some homology to the G-coupled, 7 transmembrane-spanning receptors of the prostaglandin family.
  • the receptor for prostacyclin (PGI2) is most homologous (about 70%) (See, Boie, Y. et al., J. Biol. Chem.
  • PGI 2 is a labile metabolite of arachidonic acid produced via the cyclooxygenase pathway, and plays a major physiological role as a potent mediator of vasodilation and inhibitor of platelet activation. It is primarily expressed in the kidney with lower levels of mRNA also observed in the lung and the liver. In the kidney, the PGI2 receptor is thought to play an important role in renal blood flow, renin release, and glomerular filtration rate.
  • CR3 is maximally expressed by leukocytes, placenta, testes, ovary and small intestine, and at lower levels by spleen, thymus and prostate, but not by kidney or liver. CR3, most likely thus, plays a regulatory role in cellular proliferation and or inflammation. Elevated cAMP suppresses CR3 expression early on after IL-2 stimulation, but not at a later time. Because the protein encoded by CR3 is a member of a family of 7 transmembrane spanning receptors, this receptor is likely coupled to cytoplasmic GTP- binding proteins (G-proteins), that are known to activate or suppress the generation of cAMP.
  • G-proteins cytoplasmic GTP- binding proteins
  • the CR3 gene product therefore, provides a new receptor that allows the manipulation of cellular functions controlled by biochemical pathways signaled by the receptor.
  • the CR3 gene and gene product may be used in assays for identifying other ligands that trigger the receptor. These ligands can be used to modulate cellular proliferation and inflammation.
  • DNA and polypeptide analogues and fragment analogues and original DNA and polypeptide CR3 sequences are also pan of the invention, in the form described above for CRI.
  • the CR4 gene comprises a 2946 nucleotide fragment of SEQ. ID NO: 7, which encodes a protein of 763 amino acids (about 85.9 kD), of SEQ. ID NO: 8. These sequences are shown in Table V below.
  • GGC GCC AAT CAT GTC AAT TTT GGC CAG CAA CCA GTT CCA GGG AAC ACA 1048
  • AAAAGG CCCCTT TTTTGG GGAAGG-- CCAAAA CCAAGG GGTTTT TTCCGG
  • AACCCC AAAACC AACCAA GGAAGG GGTTGG TTCCTT TTCCCC GGAAAA 1336
  • the present invention still further pertains to a CR4 polypeptide, preferably a substantially pure preparation of a CR4 polypeptide, or a recombinant CR4 polypeptide.
  • the CR4 polypeptide comprises an amino acid sequence at least about 60% to 95%, preferably about 80% to 90%, homologous to the amino acid sequence in SEQ ID NO: 8, essentially the same as the amino acid SEQ ID NO: 8, comprising at least about 5 to 150, preferably about 10 to 100. More preferably about 20 to 50 amino acids in length, comprises at least about 5 to 150, preferably at least about 10 to 100, and more preferably at least about 20 to 50 contiguous amino acids from SEQ ID NO: 8.
  • a protein homologous to SEQ ID NO: 8 has a molecular weight in the range of about 75-90 kD, preferably about 83 .
  • a polypeptide having at least one biological a ⁇ ivity of the CR4 polypeptide may differ in amino acid sequence from me sequence in SEQ ID NO:8, but such differences result in a modified polypeptide which functions in the same or similar manner as native CR4 protein or which has the same or similar characteristics of the native CR4 protein.
  • Such a peptide can include at least about 1 to 30, preferably about 2 to 20, and more preferably about 3 to 10, or about 5 amino acid residues from residues 1-763 of SEQ ID NO: 8.
  • the CR4 polypeptide is a recombinant fusion protein which comprises a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated to a protein represented by SEQ ID NO: 8, e.g., the second polypeptide portion is glutathione-S-transferase, a DNA binding domain, a polymerase a ⁇ ivating domain, e.g., the fusion protein is fun ⁇ ional in a two-hybrid assay.
  • a second polypeptide portion e.g., a second polypeptide having an amino acid sequence unrelated to a protein represented by SEQ ID NO: 8, e.g., the second polypeptide portion is glutathione-S-transferase, a DNA binding domain, a polymerase a ⁇ ivating domain, e.g., the fusion protein is fun ⁇ ional in a two-hybrid assay.
  • an immunogen comprising a CR4 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the CR4 polypeptide; e.g. a humoral response, an antibody response; or a cellular response.
  • the immunogen comprises an antigenic determinant, e.g. a unique determinant, comprising SEQ ID NO: 8.
  • a further aspect of the present invention features a monoclonal or polyclonal antibody preparation specifically reactive with an epitope of the CR4 immunogen.
  • Another aspe ⁇ of the present invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes a CR4 polypeptide, analogues and/or fragments thereof.
  • the CR4 nucleic acid comprises a transcriptional regulatory sequence, e.g., at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the CR4 gene sequence, e.g., to render the CR4 gene sequence suitable for use as an expression ve ⁇ or.
  • the nucleic acid which encodes a CR4 polypeptide of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least about 12 to 300, more preferably to at least about 20 to 200, more preferably to at least about 40 to 100 consecutive nucleotides of SEQ ID NO: 7.
  • the CR4 nucleic acid hybridizes to a nucleic acid probe corresponding to a subsequence encoding at least about 4 to 60, more preferably at least about 10 to 40, and even more preferably at least about 20 to 30 consecutive amino acid residues between residues 1-763 of SEQ ID NO: 8.
  • the nucleic acid sequence comprises at least about 1, 2, 3 or 5, and preferably at least about 10. 20, 50, or 100 nucleotides from the region of SEQ ID NO: 7 which encodes amino acid residues 1-763 of SEQ ID NO: 8.
  • the encoded peptide comprises at least about 1, 2, 3, 5, 10, 20, or 30 amino acid residues from amino acid residues 1- 763 of SEQ ID NO: 8.
  • DNA and polypeptide analogues and fragments of analogues and original DNA and polypeptide CR4 sequences are also pan of the invention, in the form described above for CRI. The sequence of this gene is identical to a gene reported as SATB-1 (Dickinson, L. A.
  • CR4 is the only CR gene also a ⁇ ivated by the TCR. In addition, elevated cAMP actually stimulates CR4 gene expression. Because the CR4 gene produ ⁇ binds to special AT-rich regions of DNA known to associate with proteins in the nuclear mating, CR4 is most likely a novel nuclear matrix protein.
  • the nuclear matrix proteins are known to influence the structure of DNA, facilitating transcription of specific genes in particular differentiated tissues.
  • CR4 can be used in an assay to identify such novel agents which can be used, for example, to treat transplant recipients by, for example, inhibiting the recipient's T cell immune response. These agents can also be used to stimulate the T cell immune response in immunosuppressed subjects, e.g., AIDS patients.
  • the CR5 gene comprises a 2020 nucleotide fragment of SEQ. ID NO: 9, which encodes a protein of 258 amino acids (about 28 kD), of SEQ. ID NO: 10. The sequences are shown in Table VI below.
  • VHL Von Hippel Lindau Disease
  • the VHL gene is thought to encode a novel tumor suppressor gene, in which persons with mutant VHL genes have a high incidence of tumors, particularly renal cell carcinomas, and pheochromocytomas. Since CR5 expression is markedly induced during IL-2-promoted T-cell proliferation, it is likely that CR5 potentiates mRNA transcript elongation, thereby antagonizing the suppressive effect of VHL.
  • a CR5 polypeptide preferably a substantially pure preparation of a CR5 polypeptide, or a recombinant CR5 polypeptide.
  • the CR5 polypeptide comprises an amino acid sequence at least about 60% to 95%, preferably about 80% to 90% homologous to the amino acid sequence in SEQ ID NO: 10, essentially the same as the SEQ ID NO: 10, at least about 5 to 150, preferably about 10 to 100, more preferably about 210 to 50 amino acids in length, comprises at least about 5 to 150, preferably at least about 10 to 100, more preferably at least about 20 to 50, contiguous amino acids from SEQ ID NO: 10.
  • a protein homologous to SEQ ID NO: 10 has a molecular weight in the range of 20-35 kD, preferably about 28 kD.
  • a polypeptide having at least one biological activity of the CR5 polypeptide may differ in amino acid sequence from the SEQ ID NO: 10, but such differences result in a modified polypeptide which functions in the same or similar manner, or has the same or similar characteristics as native CR5 protein.
  • Such a peptide can include at least about 1, 2, 3, or 5, and preferably about 10, 20, and 30, amino acid residues from residues 1-258 of SEQ ID NO: 10.
  • the CR5 polypeptide is a recombinant fusion protein which comprises a second polypeptide portion, e.g., having an amino acid sequence unrelated to a protein represented by SEQ ED NO: 10, e.g., glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain, e.g., the fusion protein is functional in a two-hybrid assay.
  • Yet another aspect of the present invention concerns an immunogen comprising a CR5 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the CR5 polypeptide; e.g. a humoral, antibody, or cellular response.
  • the immunogen comprises an antigenic determinant, e.g. a unique determinant, from a protein represented by SEQ ID NO: 10.
  • a further aspect of the present invention features a monoclonal or polyclonal antibody preparation specifically reactive with an epitope of the CR5 immunogen.
  • Another aspect of the present invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes a CR5 polypeptide, analogues and/or fragments thereof.
  • the CR5 nucleic acid comprises a transcriptional regulatory sequence, e.g., at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the CR5 gene sequence, e.g., to render the CR5 gene sequence suitable for use as an expression vector.
  • the nucleic acid which encodes an CR5 polypeptide of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least about 12 to 150, more preferably to at least about 20 to 100, and still more preferably to at least about 40 to 60 consecutive nucleotides of SEQ ID NO: 9.
  • the CR5 encoding nucleic acid hybridizes to a nucleic acid probe corresponding to a subsequence encoding at least 4 to 60 consecutive amino acids, more preferably at least about 10 to 50, and even more preferably at least about 20 to 30 amino acid residues between residues 1-258 of SEQ ID NO: 10.
  • the nucleic acid sequence comprises at least about 1, 2, 3 or 5, and preferably at least about 10, 20, 50, or 100 nucleotides from the region of SEQ ID NO: 9, which encodes amino acid residues 1-258 of SEQ ID NO: 10.
  • the encoded peptide comprises at least about 1, 2, 3, 5, 10, 20, or 30 amino acid residues from amino acid residues 1-258 of SEQ ID NO: 10.
  • DNA and polypeptide analogues and fragments of analogues and original DNA and polypeptide CR5 sequences are also part of the invention, in the form described above for CRI .
  • CR5 protein In the middle of the open reading frame of the CR5 protein is about a 100 amino acid region that has sequence homology (about 25-35%) to src homology 2 (SH2) domains (Waksman, G. et al. (1993) Cell 72: 779-790), found in many proteins that bind to phosphotyrosine residues, e.g., kinases, substrates, linking molecules, and transcription fa ⁇ ors. On either side of this SH2 domain the amino acid sequence is very rich in proline residues. Analysis of CR5 protein expression by different tissues reveals a high level of expression in heart, placenta, lung, liver skeletal muscle and kidney.
  • SH2 src homology 2
  • CR5 protein expression is induced by the proliferation-promoting cytokines IL-2, IL-3, IL-4, IL-5, but not by IL-6. Also, CR5 protein expression is induced by IFN- ⁇ and elevated intracellular cAMP, both of which antagonize IL-2 promoted proliferation.
  • CR5 protein has been found to intera ⁇ with a subunit of the RNA polymerase II preinitiation complex, termed RNA polymerase II elongation factor SHI, pl5 subunit (Garret, K.P. et al., P.N.A.S. (USA) 91: 5237-5241 (1994)).
  • RNA polymerase II elongation fa ⁇ or The pl5 subunit of this RNA polymerase II elongation fa ⁇ or is known to be responsible for promoting the elongation of transcripted mRNA molecules. Therefore, CR5 appears to fun ⁇ ion as a ligand-stimulated fa ⁇ or that facilitates mRNA expression by promoting the full elongation of mRNA transcripts. This phenomenon promises to be a novel way in which ligand-receptor systems can regularly promote gene expression. Previously, attention has focused almost entirely on this initiation of transcription, not the elongation of transcripts that were prematurely truncated. Accordingly, novel agents or drugs that modify CR5 gene expression or CR5 function have the potential to provide new ways to alter ligand-stimulated gene expression and thereby alter cellular function.
  • the CR6 gene includes a 1066 nucleotide fragment of SEQ. ID NO: 11, which encodes protein of 159 amino acids (about 17.5 kD) of SEQ. ID NO: 12. These sequences are shown in Table VII below.
  • GGG AAA GCG CTG CAT GAG TTG CTG CTG TCG GCG CAG CGT CAG GGC TGC 211 Gly Lys Ala Leu His Glu Leu Leu Leu Ser Ala Gin Arg Gin Gly Cys 25 30 35
  • the present invention further pertains to a CR6 polypeptide, preferably a substantially pure preparation of a CR6 polypeptide, or a recombinant CR6 polypeptide.
  • the CR6 polypeptide comprises an amino acid sequence at least about 60% to 95%, preferably about 80% to 90%, homologous to the amino acid sequence in SEQ ID NO: 12, essentially the same as the amino acid sequence in SEQ ID NO: 12, comprises at least about 5 to 150, preferably about 10 to 100, more preferably about 20 to 50 amino acids in length, at least about 5 to 150, preferably at least about 10 to 100, more preferably at least about 20 to 50, more preferably at least about 50, 100, or 150 contiguous amino acids from SEQ ID NO: 12.
  • a protein homologous to SEQ ID NO: 12 has a molecular weight in the range of 15-25 kD, preferably about 17 kD.
  • a polypeptide having at least one biological activity of the CR6 polypeptide may differ from the sequence in SEQ ID NO: 12, but such differences result in a modified polypeptide which functions in the same or similar manner as, or which has the same or similar characteristics as, native CR6 protein.
  • Such a peptide can comprise at least about 1 to 100, 2, 3, or 5 to 80, and preferably about 10 to 70, 20, and 30 to 50, amino acid residues from residues 1-159 of SEQ ID NO: 12.
  • the CR6 polypeptide is a recombinant fusion protein which comprises a second polypeptide portion, e.g., having an amino acid sequence unrelated to a protein represented by SEQ ID NO: 12, e.g., the second polypeptide portion is glutathione-S-transferase, a DNA binding domain, a polymerase activating domain, e.g. the fusion protein is functional in a two-hybrid assay.
  • Yet another aspect of the present invention concerns an immunogen comprising a CR6 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the CR6 polypeptide; e.g.
  • the immunogen comprises an antigenic determinant, e.g. a unique determinant, from a protein represented by SEQ ID NO: 12.
  • an antigenic determinant e.g. a unique determinant, from a protein represented by SEQ ID NO: 12.
  • a further aspect of the present invention features a monoclonal or polyclonal antibody preparation specifically reactive with an epitope of the CR6 immunogen.
  • a substantially pure nucleic acid having a nucleotide sequence which encodes a CR6 polypeptide, analogues and/or fragments thereof.
  • the CR6 nucleic acid comprises a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the CR6 gene sequence, e.g., to render the CR6 gene sequence suitable for use as an expression vector.
  • the nucleic acid which encodes an CR6 polypeptide of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least about 12 and more preferably to at least about 20, and more preferably to at least about 40 consecutive nucleotides of SEQ ID NO: 11.
  • the CR6 encoding nucleic acid hybridizes to a nucleic acid probe corresponding to a subsequence encoding at least 4 consecutive amino acids, more preferably at least about 10, and even more preferably at least about 20 amino acid residues between residues 1-159 of SEQ ID NO: 12.
  • the nucleic acid sequence comprises at least about 1, 2, 3 or 5, and preferably at least about 10, 20, 50, or 100 to 130 nucleotides from the region of SEQ ID NO: 11 which encodes amino acid residues 1-159 of SEQ ID NO: 12.
  • the encoded peptide includes at least about 1, 2, 3, 5, 10, 20, or 30 to 150 amino acid residues from amino acid residues 1-159 of SEQ ID NO: 12.
  • DNA and polypeptide analogues and fragments of analogues and original DNA and polypeptide CR6 sequences are also part of the invention, in the form described above for CRI.
  • GADD45 binds to p21, which is another p53- induced gene.
  • p21 inhibits progression through Gl of the cell cycle by binding to and suppressing cyclin-CDK complexes.
  • p21 has also been shown to bind to PCNA, preventing DNA replication. It is likely that CR6 also binds to p21, and it may facilitate cellular proliferation, by preventing the inhibitory a ⁇ ivity of p21.
  • GADD45 This gene belongs to a family of small nuclear-localizing gene produ ⁇ s. Two other members of this family, GADD45 and MyD118, have been identified. GADD45 was cloned from human fibroblasts induced by UV irradiation (Papathanasiou, M. A. et al., Moi. Cell Biol. 11(2): 1009-1016 (1991)). This protein is regulated by p53 and suppresses growth of cells by binding to PCNA, a co-factor required for DNA polymerase ⁇ a ⁇ ivity. (Smith, M. L. et al., Science 266: 1376-1380 (1994)).
  • MyD118 was cloned from M1D+ myeloid precursors following indu ⁇ ion of terminal differentiation and growth a ⁇ est by IL6. (Abdollahi, A. et al., Oncogene 6: 165-167 (1991)).
  • CR6 is about 65% homologous to GADD45.
  • CR6 is about 54% homologous to GADD45.
  • CR6 is about 66% homologous to MyD118.
  • CR6 is about 53% homologous to MyD118.
  • the CR6 protein is expressed only in testes, ovary and prostate, and its expression is suppressed by elevated cAMP.
  • the CR6 gene produ ⁇ most likely plays a role in DNA replication.
  • experiments have indicated that CR6 expression is not induced by agents that damage DNA, such as UV light.
  • CR6 does not bind to PCNA.
  • CR6 does promote DNA replication in vitro, and it is likely to be a novel CD-factor necessary for DNA replication. Therefore, the CR6 gene product can be used to identify inhibitors of DNA replication which can be used as anti-proliferative agents, e.g., in the treatment of cancer .
  • the CR7 gene includes a 2400 nucleotide DNA (SEQ. ID No: 27), encoding a protein of 313 amino acids (about 34 kD) of SEQ. ID No: 28.
  • the nucleotide (SEQ. ID No: 27), and amino acid sequence (SEQ. ID No: 28) are shown in Table VII below.
  • GAA ATC CAG AAC CAT CCA TGG ATG CAA GAT GTT CTC CTG CCC CAG GAA 908 CTT TAG GTC TTG GTA GGT ACC TAC GTT CTA CAA GAG GAC GGG GTC CTT Glu He Gin Asn His Pro Trp Met Gin Asp Val Leu Leu Pro Gin Glu 285 290 295 ACT GCT GAG ATC CAC CTC CAC AGC CTG TCG CCG GGG CCC AGC AA 952
  • GGAACAACAT TTACAACTCA TTCCAGATCC CAGGCCCCTG GAGGCTGCCT CCCAACAGTG 1140 CCTTGTTGTA AATGTTGAGT AAGGTCTAGG GTCCGGGGAC CTCCGACGGA GGGTTGTCAC GGGAAGAGTG ACTCTCCAGG GGTCCTAGGC CTCAACTCCT CCCATAGATA CTCTCTTCTT 1200 CCCTTCTCAC TGAGAGGTCC CCAGGATCCG GAGTTGAGGA GGGTATCTAT GAGAGAAGAA CTCATAGGTG TCCAGCATTG CTGGACTCTG AAATATCCCG GGGGTGGGGG GTGGGGGTGG 1260 GAGTATCCAC AGGTCGTAAC GACCTGAGAC TTTATAGGGC CCCCC CACCCCCACC GTCAGAACCC TGCCATGGAA CTGTTTCCTT CATCATGAGT TCTGCTGAAT GCCGCGATGG 1320 CAGTCTTGGG ACGGTACCTT GACAAAGGAA GTAGTACTCA
  • GTAGTATACA AAAAGATTGT AGTGGATCTA ATTTTTAAGA AATTTTGCCT TTAAGTTATT 1800 CATCATATGT TTTTCTAACA TCACCTAGAT TAAAAATTCT TTAAAACGGA AATTCAATAA TTACCTGTTT TTGTTTCTTG TTTTGAAAGA TGCGCATTCT AACCTGGAGG TCAATGTTAT 1860 AATGGACAAA AACAAAGAAC AAAACTTTCT ACGCGTAAGA TTGGACCTCC AGTTACAATA GTATTTATTT ATTTATTTAT TTGGTTCCCT TCCTATTCCA AGCTTCGCTG CTGCCCTAGT 1920 CATAAATAAA TAAATAAATA AACCAAGGGA AGGATAAGGT TCGAAGCGAC GACGGGATCA TTTCTTTCCT CCTTTCCTCC TCTGACTTGG GGACCTTG GGGGAGGGCT GCGACGCTTG 1980 AAAGAAAGGA GGAAAGGAGG AGACTGAACC CCTGGAAAAC CCTCCCTC
  • CR7 is an IL-2-induced gene and is a serine/threonine-specific protein kinase involved in T cell lymphomagenesis.
  • the CR7 gene turned out to be identical to the putative proto-oncogene, pim-1, which has been reported to be over-expressed in about 50% of Moloney murine leukemia virus (MuLV)-induced T cell lymphomas (See, Selten, G. et al., Cell 46: 603-611 (1986)).
  • MoLV Moloney murine leukemia virus
  • the present invention further pertains to a CR7 polypeptide, preferably a substantially pure preparation of a CR7 polypeptide, or a recombinant CR7 polypeptide.
  • the CR7 polypeptide comprises an amino acid sequence at least about 60% to 95%, preferably about 80% to 90%, homologous to the amino acid sequence in SEQ ID NO: 28; essentially the same as the amino acid sequence in SEQ ID NO: 28, at least about 5 to 150, preferably about 10 to 100, more preferably about 20 to 50 amino acids in length, at least about 5, preferably at least about 10, more preferably at least about 20, more preferably at least about 50, 100, or 150 contiguous amino acids from SEQ ID NO: 28.
  • a protein homologous to SEQ ID NO: 28 has a molecular weight in the range of about 15-25 kD, preferably about 17 kD.
  • a polypeptide having at least one biological activity of the CR7 polypeptide may differ in amino acid sequence from the sequence in SEQ ID NO: 28, but such differences result in a modified polypeptide which functions in the same or similar manner as or which has the same or similar characteristics as native CR7 protein.
  • Such a peptide can comprise at least about 1, 2, 3, or 5 to 140, and preferably about 10, 20, and 30 to 100, amino acid residues from residues 1-159 of SEQ ED NO: 28.
  • the CR7 polypeptide is a recombinant fusion protein which comprises a second polypeptide portion, e.g., having an amino acid sequence unrelated to a protein represented by SEQ ID NO: 28, e.g., the second polypeptide portion is glutathione-S-transferase, a DNA binding domain, a polymerase activating domain, e.g. the fusion protein is functional in a two-hybrid assay.
  • Yet another aspect of the present invention concerns an immunogen comprising a CR7 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the CR7 polypeptide; e.g.
  • the immunogen comprises an antigenic determinant, e.g. a unique determinant, from a protein represented by SEQ ID NO: 28.
  • a further aspect of the present invention features a monoclonal or polyclonal antibody preparation specifically reactive with an epitope of the CR7 immunogen.
  • nucleic acid having a nucleotide sequence which encodes a CR7 polypeptide, analogues and/or fragments thereof.
  • the CR7 nucleic acid comprises a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the CR7 gene sequence, e.g., to render the CR7 gene sequence suitable for use as an expression vector.
  • the nucleic acid which encodes an CR7 polypeptide of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least about 12 to 300, and more preferably to at least about 20 to 200, and more preferably to at least about 40 to 100 consecutive nucleotides of SEQ ID NO: 27.
  • the CR7 encoding nucleic acid hybridizes to a nucleic acid probe corresponding to a subsequence encoding at least about 4 consecutive amino acids, more preferably at least about 10, and even more preferably at least about 20 amino acid residues between residues 1-159 of SEQ ID NO: 28.
  • the nucleic acid sequence comprises at least about 1, 2, 3 or 5, and preferably at least about 10, 20, 50, or 100 to 400 nucleotides from the region of SEQ ID NO: 27 which encodes amino acid residues 1-159 of SEQ ID NO: 28.
  • the encoded peptide includes at least about 1, 2, 3, 5, 10, 20, or 30 to 100 amino acid residues from amino acid residues 1-159 of SEQ ID NO: 28.
  • DNA and polypeptide analogues and fragments of analogues and original DNA and polypeptide CR7 sequences are also part of the invention, in the form described above for CRI.
  • the CR8 gene includes a 2980 nucleotide fragment of SEQ. ID NO: 13, which encodes (via a 3.2 Kb mRNA transcript) a protein of 412 amino acids (about 45 kD) of SEQ. ID NO: 14. These sequences are shown in Table VIII below.
  • CAG CAG CAG CAG CAG AAA ATC ATT GCC CTG CAG AGT GGT TTA CAA GCT GGT 623
  • GAG AAC ACT CGG GAC CTG AAG TCT TCG CAG CTT GTC ACC CAC CTC CAC 767
  • the present invention still further pertains to a CR8 polypeptide, preferably a substantially pure preparation of a CR8 polypeptide, or a recombinant CR8 polypeptide.
  • the CR8 polypeptide comprises an amino acid sequence at least about 60%), to 95%, preferably about 80% to 90% homologous to the amino acid sequence in SEQ ED NO: 14, essentially the same as the amino acid sequence in SEQ ID NO: 14, comprises about 5 to 150, preferably about 10 to 100, more preferably about 20 to 50 amino acids in length, about 5 to 300, preferably at least about 10 to 200, more preferably at least about 20 to 100, more preferably at least about 50, 100, or 150 to 350 contiguous amino acids from SEQ ID NO: 14.
  • a protein homologous to SEQ ED NO: 14 has a molecular weight in the range of about 35-50 kD, preferably about 45 kD.
  • a polypeptide having at least one biological activity of the CR8 polypeptide may differ in amino acid sequence from the sequence in SEQ ID NO: 14, but such differences result in a modified polypeptide which functions in the same or similar manner as or which has the same or similar characteristics as native CR8 protein.
  • Such a peptide can comprise at least about 1, 2, 3, or 5, and preferably about 10, 20, and 30, amino acid residues from residues 1-412 of SEQ ED NO: 14.
  • the CR8 polypeptide is a recombinant fusion protein which includes a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated to a protein represented by SEQ ED NO: 14, e.g., the second polypeptide portion is glutathione-S-transferase, a DNA binding domain, a polymerase activating domain, e.g., the fusion protein is functional in a two-hybrid assay.
  • a second polypeptide portion e.g., a second polypeptide having an amino acid sequence unrelated to a protein represented by SEQ ED NO: 14, e.g., the second polypeptide portion is glutathione-S-transferase, a DNA binding domain, a polymerase activating domain, e.g., the fusion protein is functional in a two-hybrid assay.
  • an immunogen comprising a CR8 polypeptide in an immunogenic preparation, the immuno
  • the immunogen comprises an antigenic determinant, e.g. a unique determinant, from a protein represented by SEQ ID NO: 14.
  • a further aspect of the present invention features a monoclonal or polyclonal antibody preparation specifically reactive with an epitope of the CR8 immunogen.
  • Another aspect of the present invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes a CR8 polypeptide, analogues and/or fragments thereof.
  • the subject CR8 nucleic acid includes a transcriptional regulatory sequence, e.g., at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the CR8 gene sequence, e.g., to render the CR8 gene sequence suitable for use as an expression vector.
  • the nucleic acid which encodes a CR8 polypeptide of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least about 12 to 1,000, more preferably to at least about 20 to 500, more preferably to at least about 40 to 300consecutive nucleotides of SEQ ED NO: 13.
  • the CR8 encoding nucleic acid hybridizes to a nucleic acid probe corresponding to a subsequence encoding at least about 4 to 300, more preferably at least about 10 to 200, and even more preferably at least about 20 to 100 and longer amino acid residues between residues 1-412 of SEQ ID NO: 14.
  • the nucleic acid sequence includes at least about 1, 2, 3 or 5, and preferably at least about 10, 20, 50, or 100 to 1,000 nucleotides from the region of SEQ ED NO: 13 which encodes amino acid residues 1-412 of SEQ ED NO: 14.
  • the encoded peptide includes at least about 1, 2, 3, 5, 10, 20, or 30 to 350 amino acid residues from amino acid residues 1-412 of SEQ ID NO: 14.
  • DNA and peptide analogues and fragments of analogues and original DNA and polypeptide CR8 sequences are also part of the invention, in the form described above for CRI.
  • the protein encoded by the bHLH region of the gene has been expressed in E. coli and has been found to bind to a hexanucleotide predi ⁇ ed by the binding specificity of other bHLH proteins. (See Feder, J. et al., Moi. Cell Biol. 13(1): 105-113 (1993)).
  • the N-terminal basic region binds to DNA and the HLH region serves as a protein dimerization motif. From the sequence of the bHLH region, CR8 fits into a class by itself. It shares most homology with Drosophila transcription repressors of the hairy family. However, CR8 lacks amino acid residues in the basic region and a C-terminal WRPW motif, characteristic for hairy proteins. CR8 also binds to Class B E-box sites (CACGTC/CATGTG), as do the c-myc family of bHLH proteins, rather than to Class C sites (CAGCCG) preferred by hairy-related family members. CR8 is ubiquitously expressed in all tissues examined except placenta.
  • CR8 contains a bHLH domain, it is most likely a protein that binds to DNA and modifies gene expression, either by a ⁇ ivation or by suppression. Since CR8 binds to class B E-bax sequences, which the proto-oncogene c-myc family members also bind, it is likely that CR8 modifies the expression of genes important for the intermediate and late phases of ligand-promoted cell cycle progression.
  • CR8 is a prime candidate for the development of new assays to discover agents that modify cellular function by either enhancing or suppressing CR8 gene expression or CR8 fun ⁇ ion.
  • the CR8 gene and its gene produ ⁇ are described in further detail below in Example VII.
  • two are DNA binding proteins, one is a newly recognized transmembrane receptor, one contains an SH2 domain, one has homology to a newly recognized family of small proteins that regulate cellular proliferation, and another is a serine/threonine kinase known to be IL-2-induced, and to be over-expressed in MuLV-induced T cell lymphomas. Allowing for redundancies, a conservative estimate is that there are still about 40 to 50 novel genes induced by IL-2, which can be isolated using the method of the present invention.
  • the present invention pertains to an isolated nucleic acid comprising all redundant nucleotide sequences encoding the CR proteins, homologues, analogues and muteins described below.
  • the invention comprises nucleotide sequence encoding anyone of the CR proteins, e.g., CRI, CR2, CR3, CR4, CR5, CR6, CR7, and CR8, particularly those corresponding to CRI, CR2, CR3, CR5, CR6, and CR8 and/or equivalents, homologues, analogues, and fragments of such nucleic acids and of CR4 and CR7.
  • nucleic acid as used herein is intended to include fragments and equivalents.
  • equivalent refers to nucleotide sequences encoding fun ⁇ ionally equivalent CR proteins or functionally equivalent peptides which retain other activities of an CR protein such as described herein.
  • Equivalent nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants and include, therefore, sequences that differ from the nucleotide. sequence CR proteins shown in any of SEQ. ID Nos: 2, 4, 6, 8, 10, 12, 14, and 28, due to the degeneracy of the genetic code.
  • Equivalents also include nucleotide sequences that hybridize under stringent conditions (i.e., equivalent to about 20-27°C below the melting temperature (T m ) of the DNA duplex formed in about IM salt) to the nucleotide sequence encoding the presently claimed CR proteins represented as SEQ. ID Nos: 2, 4, 6, 8, 10, 12, 14, and 28.
  • equivalents further include nucleic acid sequences derived from and evolutionarily related to, any of the coding fragments of nucleotide sequences shown as SEQ. ID Nos: 1, 3, 5, 7, 9, 11, 13, and 27.
  • homologs of the subje ⁇ CR proteins which have at least one biological a ⁇ ivity of a CR protein.
  • Such homologs of the subje ⁇ CR proteins can be generated by mutagenesis, such as by discrete point mutation(s) or by truncation. For instance, mutation can give rise to homologs which retain substantially the same, or merely a subset, of the biological a ⁇ ivity of the CR protein from which it was derived.
  • antagonistic forms of the protein can be generated which are able to inhibit the fun ⁇ ion of the naturally occurring form of the CR protein.
  • a protein has CR biological activity if it has one or more of the following properties: (1) its expression is regulated by ligand-receptor stimulation; and (2) it participates in ligand-receptor modification of cellular function, e.g. proliferation, differentiation, programmed cell death.
  • the term “gene” or “recombinant gene” refers to a nucleic acid comprising an open reading frame encoding a CR protein of the present invention, including both exon and (optionally) intron sequences.
  • a “recombinant gene” refers to nucleic acid encoding a CR protein and comprising CR encoding exon sequences, though it may optionally include intron sequences which are either derived from a chromosomal CR gene or from an unrelated chromosomal gene.
  • the term “intron” refers to a DNA sequence present in a given CR gene which is not translated into protein and is generally found between exons.
  • transfe ⁇ ion means the introdu ⁇ ion of a nucleic acid, e.g., an expression ve ⁇ or, into a recipient cell by nucleic acid-mediated gene transfer.
  • Transformation refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of the CR protein of the present invention or where anti-sense expression occurs from the transferred gene, the expression of a naturally-occurring form of the CR protein is disrupted.
  • ve ⁇ or refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • ve ⁇ or is an episome, i.e., a nucleic acid capable of extra- chromosomal replication.
  • Preferred ve ⁇ ors are those capable of autonomous replication and/expression of nucleic acids to which they are linked. Ve ⁇ ors capable of dire ⁇ ing the expression of genes to which they are operatively linked are referred to herein as "expression ve ⁇ ors”.
  • expression ve ⁇ ors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer to circular double stranded DNA loops which, in their ve ⁇ or form are not bound to the chromosome.
  • plasmid and ve ⁇ or are used interchangeably as the plasmid is the most commonly used form of ve ⁇ or.
  • the invention is intended to include such other forms of expression ve ⁇ ors which serve equivalent fun ⁇ ions and which become known in the an subsequently hereto.
  • Transcriptional regulatory sequence is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked.
  • transcription of a recombinant CR gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell-type in which expression is intended.
  • the recombinant gene can also be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring form of the CR proteins.
  • tissue-specific promoter means a DNA sequence that serves as a promoter, i.e., regulates expression of a sele ⁇ ed DNA sequence operably linked to the promoter, and which effects expression of the sele ⁇ ed DNA sequence in specific cells of a tissue.
  • the term also covers so-called “leaky” promoters, which regulate expression of a sele ⁇ ed DNA primarily in one tissue, but cause expression in other tissues as well.
  • a transgenic animal is any animal, preferably a non-human mammal, e.g. a rat, a mouse or pig, in which one or more of the cells of the animal includes a transgene.
  • the transgene is introduced into the cell, directly or indirectly by introdu ⁇ ion into a precursor of the cell, by way of deliberate genetic manipulation, such as by micro inje ⁇ ion or by infe ⁇ ion with a recombinant virus.
  • the language “genetic manipulation” does not include classical cross ⁇ breeding, or in vitro fertilization, but rather is dire ⁇ ed to the introdu ⁇ ion of a recombinant DNA molecule.
  • This molecule can be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
  • the transgene causes cells to express a recombinant form of one or more of the subject CR proteins, or alternatively, .to disrupt expression of one or more of the naturally-occurring forms of the CR genes.
  • transgene refers to a nucleic acid sequence which is partly or entirely heterologous, i.e., foreign, to the animal or cell into which it is introduced, or, is homologous to an endogenous gene of the animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).
  • a transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.
  • genes for a particular polypeptide may exist in single or multiple copies within the genome of an individual. Such duplicate genes may be identical or may have certain modifications, including nucleotide substitutions, additions or deletions, which all still code for polypeptides having substantially the same a ⁇ ivity.
  • the term "DNA sequence encoding a CR protein” refers to one or more genes within a particular individual. Moreover, certain differences in nucleotide sequences may exist between individual organisms, which are called alleles. Such allelic differences may or may not result in differences in amino acid sequence of the encoded polypeptide yet still encode a protein with the same biological a ⁇ ivity.
  • Cells "host cells” or “recombinant host cells” are terms used interchangeably herein.
  • a “chimeric protein” or “fusion protein” is a fusion of a first amino acid sequence encoding one of the subject CR proteins with a second amino acid sequence defining a domain foreign to and not substantially homologous with any domain of the subject CR protein.
  • A- chimeric protein may present a foreign domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it may be an "interspecies", “intergeneric”, etc. fusion of protein stru ⁇ ures expressed by different kinds of organisms.
  • the language “evolutionarily related to”, with respect to nucleic acid sequences encoding CR proteins refers to nucleic acid sequences which have arisen naturally in an organism, including naturally occurring mutants.
  • This language also refers to nucleic acid sequences which, while derived from a naturally occurring CR nucleic, have been altered by mutagenesis, as for example, combinatorial mutagenesis, yet still encode polypeptides which have at least one a ⁇ ivity of a CR protein.
  • the present invention provides a substantially pure polynucleotide comprising a nucleic acid encoding a polypeptide having one or more of the chara ⁇ eristics and/or run ⁇ ionalities of anyone of the CR proteins, including CRI, CR2, CR3, CR4, CR5, CR6, CR7 and CR8 proteins, analogues, homologues, fragments, and combinationes thereof.
  • the nucleic acid comprises a cDNA encoding a peptide having at least one a ⁇ ivity of a subje ⁇ CR proteins.
  • the nucleic acid comprises a DNA molecule comprising at least a portion of anyone of the nucleotides SEQ.
  • Preferred portions of these cDNA molecules include the coding regions of the genes, fragments thereof, and combinations thereof.
  • Preferred nucleic acids encode a CR protein comprising an amino acid sequence about 60% to 99% homologous, preferably at least about 60% homologous, more preferably about 70% homologous and most preferably about 80%, 90%, or 95% homologous to one of anyone of the amino acid sequences SEQ. ID Nos: 2, 4, 6, 8, 10, 12, 14, or 28, or combinations or fragments thereof.
  • Nucleic acids which encode polypeptides having an activity of a subje ⁇ CR protein and having at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homology to anyone SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, or 28, are also within the scope of the invention.
  • the term "homology" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a fun ⁇ ion of the number of matching or homologous positions shared by the sequences.
  • Another aspe ⁇ of the invention provides a nucleic acid which hybridizes under high or low stringency conditions to a nucleic acid which encodes a peptide having all or a portion of amino acid SEQ. ID NO: 2, SEQ. ID NO: 4, SEQ. ID NO: 6, SEQ. ID NO: 8, SEQ. ID NO:
  • SEQ. ID NO: 12 SEQ. ID NO: 14, or SEQ. ID No: 28.
  • Appropriate stringency conditions which promote DNA hybridization for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y., 6.3.1-6.3.6 (1989).
  • the salt concentration in the wash step can be sele ⁇ ed from a low stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2 x SSC at 50°C.
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C.
  • nucleic acids encode functionally equivalent peptides , i.e., a peptide having a biological activity of a CR protein, but differ in sequence from the sequence shown in said sequence listings due to degeneracy in the genetic code. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC each encode histidine) may result in "silent" mutations which do not affect the amino acid sequence of the CR protein.
  • DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject CR proteins will exist among vertebrates.
  • these variations in one or more nucleotides (up to about 3 to 5% of the nucleotides) of the nucleic acids encoding polypeptides having an activity of an CR protein may exist among individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention. Fragments of the nucleic acids encoding the a ⁇ ive portion of the presently claimed CR proteins are also within the scope of the invention.
  • a fragment of the nucleic acid encoding the a ⁇ ive portion of a CR protein refers to a nucleic acid having fewer nucleotides than the nucleotide sequence encoding the entire amino acid sequence of a CR protein but which nevertheless encodes a peptide having a biological a ⁇ ivity of the CR proteins described herein.
  • Nucleic acid fragments within the scope of the present invention include those capable of hybridizing under high or low stringency conditions with nucleic acids from other species for use in screening protocols to dete ⁇ CR homologs, as well as those capable of hybridizing with nucleic acids from human specimens for use in dete ⁇ ing the presence of a nucleic acid encoding one of the subje ⁇ CR proteins, including alternate isoforms, e.g. mRNA splicing variants. Nucleic acids within the scope of the invention can also contain linker sequences, modified restriction endonuclease sites and other sequences useful for molecular cloning, expression or purification of recombinant forms of the subject CR proteins.
  • a nucleic acid encoding a peptide having an a ⁇ ivity of an CR protein can be obtained from mRNA present in any of a number of eukaryotic cells. Nucleic acids encoding CR proteins of the present invention can also be obtained from genomic DNA obtained from both adults and embryos. For example, a gene encoding a CR protein can be cloned from either a cDNA or a genomic library in accordance with protocols herein described, as well as those generally known to persons skilled in the art.
  • a cDNA encoding one of d e subject CR proteins can be obtained by isolating total mRNA from a cell, e.g. a mammalian cell, e.g. a human cell, including tumor cells.
  • Double stranded cDNAs can then be prepared from the total mRNA, and subsequently inserted into a suitable plasmid or ba ⁇ eriophage ve ⁇ or using any one of a number of known techniques.
  • the gene encoding the CR protein can also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention.
  • the nucleic acid of the invention may be a DNA or an RNA.
  • Preferred nucleic acids are the DNAs represented by the sequences shown in SEQ. ID Nos: 1, 3, 5, 9, 11, 13, and homologues thereof and of SEQ. ID Nos: 7 and 27, and coding fragments thereof.
  • This invention also provides expression vectors containing a nucleic acid encoding a peptide having an a ⁇ ivity of an CR protein, operably linked to at least one transcriptional regulatory sequence.
  • operably linked refers to linkage of the nucleotide sequence to a regulatory sequence in a manner which allows expression of the nucleotide sequence.
  • Regulatory sequences are art-recognized and are sele ⁇ ed to direct expression of the peptide having an a ⁇ ivity of a CR protein.
  • transcriptional regulatory sequence includes promoters, enhancers and other expression control elements.
  • the expression vector includes a recombinant gene encoding a peptide having an a ⁇ ivity of a subje ⁇ CR protein.
  • Such expression ve ⁇ ors can be used to transfe ⁇ cells and thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.
  • such ve ⁇ ors can be used as a part of a gene therapy protocol to reconstitute the function of, or alternatively, abrogate the fun ⁇ ion of one of the subject CR proteins in a cell in which a CR protein is misexpressed.
  • Another aspe ⁇ of the present invention concerns recombinant forms of the subject CR proteins which are encoded by genes derived from eukaryotic organisms, e.g. mammals, e.g. humans, and which have at least one biological activity of a CR protein.
  • recombinant protein refers to a protein of the present invention which is produced by recombinant DNA techniques, wherein generally DNA encoding the subje ⁇ CR protein is inserted into a suitable expression ve ⁇ or which is in turn used to transform a host cell to produce the heterologous protein.
  • the phrase "derived from”, with respe ⁇ to a recombinant gene encoding the recombinant CR protein includes within the meaning of "recombinant protein” those proteins having an amino acid sequence of a native CR protein of the present invention, or an amino acid sequence similar thereto which is generated by mutations including substitutions and deletions (including truncation) of a naturally occurring CR protein of a organism.
  • Preferred recombinant proteins in accordance with the present invention, in addition to native CR proteins are those about 60 to 99% homologous, preferably at least about 60% homologous, more preferably at least about 70% homologous and most preferably at least about 80% homologous to any one of amino acid SEQ. ID Nos: 2, 4,.
  • Polypeptides having an activity of the subje ⁇ CR proteins and having about 60% to 99% homology, preferably at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homology to anyone of SEQ. ID Nos.: 2, 4, 6, 8, 10, 12, 14, or 28, are also within the scope of the invention.
  • the present invention further pertains to recombinant forms of the subje ⁇ CR proteins which are encoded by genes derived from an organism and which have amino acid sequences evolutionarily related to a CR protein of either SEQ. ID NO: 2, 4, 6, 8, 10, 12, or 14.
  • the language "evolutionarily related to,” with respe ⁇ to amino acid sequences of the present recombinant CR proteins, refers to CR proteins having amino acid sequences which have arisen naturally, as well as mutational variants of CR proteins which are derived, for example, by combinatorial mutagenesis.
  • Preferred evolutionarily derived CR proteins are at least 60% homologous, more preferably at least about 70% homologous and most preferably at least about 80% homologous to anyone of SEQ. ID NO: 2, SEQ. ID NO: 4, SEQ. ID NO: 6, SEQ. ID NO: 8, SEQ. ID NO: 10, SEQ. ID NO: 12, SEQ. ID NO: 14, or SEQ. ID NO: 28.
  • Polypeptides having about 60 to 99% homology, preferably at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homology with a sequence shown in any of SEQ ID Nos: 2, 4, 6, 8, 10, 12, or 14 are also within the scope of the invention.
  • the present invention further pertains to methods of producing the subje ⁇ CR proteins.
  • a host cell transfe ⁇ ed with a nucleic acid vector dire ⁇ ing expression of a nucleotide sequence encoding the subject CR protein can be cultured under appropriate conditions to allow expression of the peptide to occur.
  • the peptide can be secreted and isolated from a mixture of cells and medium containing the recombinant CR peptide.
  • the peptide can be retained cytoplasmically and the cells harvested, lysed and the protein isolated.
  • a cell culture includes host cells, media and other byprodu ⁇ s. Suitable media for cell culture are well known in the art.
  • the recombinant CR peptide may be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, ele ⁇ rophoresis, and immunoaffinity purification with antibodies specific for such peptide.
  • the recombinant CR protein is a fusion protein containing a domain which facilitates its purification.
  • This invention also pertains to a host cell transfe ⁇ ed to express a recombinant form of at least one of the subje ⁇ CR proteins.
  • the host cell can be any prokaryotic or eukaryotic cell.
  • a nucleotide sequence derived from the cloning of the CR proteins of the present invention, encoding all or a sele ⁇ ed portion of a protein can be used to produce a recombinant form of a CR protein via microbial or eukaryotic cellular processes.
  • Ligating the polynucleotide sequence into a gene constru ⁇ such as an expression ve ⁇ or, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, inse ⁇ or mammalian) or prokaryotic (ba ⁇ erial cells), are standard procedures used in producing other well-known proteins, e.g. insulin, interferons, human growth hormone, IL-1, IL-2, and the like. Similar procedures, or modifications thereof, can be employed to. prepare recombinant CR proteins, or portions thereof, by microbial means or tissue-culture technology in accordance with the subje ⁇ invention.
  • a recombinant CR gene may be produced by ligating a nucleic acid encoding anyone of thet CR proteins, or fragments thereof, into a ve ⁇ or suitable for expression in either prokaryotic cells, eukaryotic cells, or both.
  • Expression ve ⁇ ors for produ ⁇ ion of recombinant forms of the subje ⁇ CR proteins include plasmids and other ve ⁇ ors.
  • suitable ve ⁇ ors for the expression of a CR protein include plasmids of the types: pBR322-derived plasmids, pEMBL- derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.
  • plasmids of the types: pBR322-derived plasmids, pEMBL- derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.
  • ve ⁇ ors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid.
  • drug resistance markers such as ampicillin can be used.
  • Preferred mammalian expression ve ⁇ ors contain both prokaryotic sequences to facilitate the propagation of the vector in ba ⁇ eria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.
  • the pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived ve ⁇ ors are examples of mammalian expression ve ⁇ ors suitable for transfection of eukaryotic cells. Some of these ve ⁇ ors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance sele ⁇ ion in both prokaryotic and eukaryotic cells.
  • viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells.
  • BBV-1 bovine papilloma virus
  • pHEBo Epstein-Barr virus
  • pHEBo Epstein-Barr virus
  • pREP-derived and p205 Epstein-Barr virus
  • the recombinant CR protein can be expressed using a baculovirus expression system.
  • baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived ve ⁇ ors (such as the ⁇ -gal containing pBlueBac III).
  • a start codon AGT
  • methionine aminopeptidase MAP
  • the coding sequences for the polypeptide can be incorporated as a part of a fusion gene including a nucleotide sequence encoding a different polypeptide.
  • This type of expression system can be useful under conditions where it is desirable to produce an immunogenic fragment of a CR protein.
  • the nucleic acid sequences corresponding to the portion of a subject CR protein to which antibodies are to be raised can be incorporated into a fusion gene constru ⁇ which includes coding sequences for a late vaccinia virus structural protein to produce a set of recombinant viruses expressing fusion proteins comprising a portion of the CR protein as part of the virion.
  • the Multiple Antigen Peptide system for peptide-based immunization may also be utilized to generate an immunogen, wherein a desired portion of a subje ⁇ CR protein is obtained dire ⁇ ly from organo-chemical synthesis of the peptide onto an oligomeric branching lysine core (see, for example, Posnett et al., J.B.C. 263: 1719 (1988), and Nardelli et al., J. Immunol. 148: 914 (1992)).
  • Antigenic determinants of me subje ⁇ CR proteins can also be expressed and presented by bacterial cells.
  • fusion proteins can also facilitate the expression of proteins, such as any one of the CR proteins of the present invention.
  • a CR protein of the present invention can be generated as a glutathione-S-transferase (GST- fusion protein).
  • GST fusion proteins can enable easy purification of the CR protein, such as by the use of glutathione-derivatized matrices (see, for example, Current Protocols in Molecular Biology, eds. Ausabel et al., N. Y., John Wiley & Sons (1991)). Techniques for making fusion genes are known to those skilled in the art.
  • the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restri ⁇ ion enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, Eds. Ausubel et al., John Wiley & Sons (1992)).
  • a biological a ⁇ ivity of a CR protein includes: promotion of cell cycle progression (e.g., CRI); ligand-receptor signaling (e.g., CR3); cellular maturation, differentiation, and proliferation (e.g., CR4); enhancement or suppression of DNA replication (e.g., CR5, CR6); promotion of mRNA transcription by stimulating elongation of mRNA transcription (e.g., CR5, CR6); and transcriptional a ⁇ ivation and repression (e.g., CR8).
  • CRI cell cycle progression
  • ligand-receptor signaling e.g., CR3
  • cellular maturation, differentiation, and proliferation e.g., CR4
  • enhancement or suppression of DNA replication e.g., CR5, CR6
  • promotion of mRNA transcription by stimulating elongation of mRNA transcription e.g., CR5, CR6
  • transcriptional a ⁇ ivation and repression e.g.
  • a polypeptide having at least one biological a ⁇ ivity of the subject CR proteins may differ in amino acid sequence from the sequence shown in either SEQ. ID NO: 2, 4, 6, 8, 10, 12, or 14, but such differences result in a modified polypeptide which fun ⁇ ions in the same or similar manner as the native CR or which has the same or similar chara ⁇ eristics of the native CR protein.
  • Various modifications of a CR protein of the present invention to produce these and other fun ⁇ ionally equivalent peptides are described in detail herein.
  • the terms peptide, protein, and polypeptide are used interchangeably herein.
  • the present invention also pertains to isolated CR proteins which are isolated from, or otherwise substantially free of other cellular proteins normally associated with the CR protein.
  • the language “substantially free of other cellular proteins” also referred to herein as "contaminating proteins" or “substantially pure, substantially pure preparation, or purified preparations” are defined as encompassing CR protein preparations having less than about 20% (by dry weight) contaminating protein, and preferably having less than about 5% contaminating protein.
  • Fun ⁇ ional forms of the subje ⁇ CR proteins can be prepared, for the first time, as purified preparations by using a cloned gene as described herein.
  • the term “purified” as used herein preferably means at least about 80% by dry weight, more preferably in the range of about 95 to 99% by weight, and most preferably at least about 99.8% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present).
  • the term “pure” as used herein preferably has the same numerical limits as the term “purified”.
  • isolated and purified do not encompass either natural materials in their native state or natural materials that have been separated into components (e.g., in an acrylamide gel) but not obtained either as pure (e.g. lacking contaminating proteins, or chromatography reagents such as denaturing agents and polymers, e.g. acrylamide or agarose) substances or solutions.
  • isolated as also used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, respe ⁇ ively, that are present in the natural source of the macromolecule.
  • an isolated nucleic acid encoding one of the subject CR proteins preferably includes no more than 10 Kilobases (Kb) of nucleic acid sequence which naturally immediately flanks that particular CR gene in genomic DNA, more preferably no more than 5 Kb of such namrally occurring flanking sequences, and most preferably less than 1.5 Kb of such naturally occurring flanking sequence.
  • Kb Kilobases
  • isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • an "isolated nucleic acid” is meant to include nucleic acid fragments which are not namrally occurring as fragments and would not be found in the natural state.
  • isolated peptide portions of the subject CR proteins can also be obtained by screening peptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such peptides.
  • fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
  • a CR protein of the present invention can be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length.
  • the fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments which can fun ⁇ ion as either agonists or antagonists of a CR protein a ⁇ ivity, such as by microinje ⁇ ion assays.
  • the structure of the subject CR proteins may be modified for such purposes as enhancing therapeutic or prophyla ⁇ ic efficacy, or stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo).
  • modified peptides when designed to retain at least one activity of the naturally-occurring form of the protein, are considered fun ⁇ ional equivalents of the CR proteins described in more detail herein.
  • modified peptide can be produced, for instance, by amino acid substitution, deletion, or addition.
  • Whether a change in the amino acid sequence of a peptide results in a functional CR homolog can be readily determined by assessing the ability of the variant peptide to produce a response in cells in a fashion similar to the wild-type CR protein or peptide. Peptides in which more than one replacement has taken place can readily be tested in the same manner.
  • Another aspect of the invention pertains to an antibody or antibody preparation specifically rea ⁇ ive with at least one epitope of at least one of the subject CR proteins.
  • immunogens derived from the present CR proteins based on the cDNA sequences
  • anti-protein anti-peptide antisera or monoclonal antibodies can be made by standard protocols (see, for example, Antibodies: A Laboratory Manual, Ed., Harlow and Lane (Cold Spring Harbor Press: 1988)).
  • a mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide, e.g., CR protein or an antigenic fragment which is capable of eliciting an antibody response.
  • Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or odier techniques well known in the art.
  • An immunogenic portion of the subject CR proteins can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
  • the subject antibodies are immunospecific for antigenic determinants of the CR proteins of the present invention, e.g. antigenic determinants of a protein represented by one of SEQ.
  • the anti-CR protein antibodies do not substantially cross react, i.e., rea ⁇ specifically, with a protein which is, e.g., less than about 90% homologous to one of SEQ. ID Nos: 2, 4, 6, 8, 10, 12, 14, or 28, e.g., less than about 95% homologous to one of SEQ. ID Nos: 2, 4, 6, 8, 10, 12, 14, or 28, e.g., less than about 98 to 99% homologous to one of SEQ.
  • the language "not substantially cross-rea ⁇ ” means that the antibody has a binding affinity for a non-homologous protein which is less than about 10%, more preferably less than about 5%, and even more preferably less than about 1 %, of the binding affinity for a protein of SEQ. ID Nos: 2, 4, 6, 8, 10, 12, 14, or 28.
  • anti-CR antisera may be obtained and, if desired, polyclonal anti-CR antibodies isolated from the serum.
  • antibody producing cells lymphocytes
  • myeloma cells to yield hybridoma cells.
  • Hybridoma cells can be screened immunochemically for produ ⁇ ion of antibodies specifically rea ⁇ ive with a CR protein of the present invention and monoclonal antibodies isolated from a culture comprising such hybridoma cells.
  • antibody as used herein is intended to include fragments thereof which are also specifically rea ⁇ ive with one of the subject CR proteins.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments.
  • Antibodies of the present invention are further intended to include bi-specific and chimeric molecules having an anti-CR portion.
  • Both monoclonal and polyclonal antibodies (Ab) dire ⁇ ed against CR or CR variants, and antibody fragments such as Fab' and F(ab')2, can be used to block the action of a CR proteins and allow the study of the role of the particular CR protein of the present invention in cell signaling.
  • the nucleotide sequence determined from the cloned cDNA of the CR proteins from a human cell line permits the preparation of probes for identification of CR homologues in other human cell types, as well as CR homologues from other animals.
  • the present invention also provides a probe/primer comprising a substantially purified oligonucleotide, wherein the oligonucleotide comprises a region of nucleotide sequence which hybridizes under stringent conditions to at least about 10 consecutive nucleotides of sense or anti-sensenucleic acids of SEQ. ID Nos: 1, 3, 5, 7, 9, 11, 13, or 27, or naturally occurring mutants thereof.
  • the probe/primer further comprises a label group attached thereto, which may be dete ⁇ ed, e.g., the label group may be sele ⁇ ed from radioisotopes, fluorescent compounds, enzymes, and enzyme co-fa ⁇ ors.
  • a label group attached thereto, which may be dete ⁇ ed, e.g., the label group may be sele ⁇ ed from radioisotopes, fluorescent compounds, enzymes, and enzyme co-fa ⁇ ors.
  • Such probes can be used as a part of a test kit for measuring a level of an CR nucleic acid in a sample of cells from a patient, e.g., measuring a CR mRNA level; e.g. determining whether a genomic CR gene has been mutated or deleted.
  • nucleotide probes can be generated from the cloned sequence of the subje ⁇ CR proteins, which allow for histological screening of inta ⁇ tissue and tissue samples for the presence of a CR mRNA.
  • Use of probes dire ⁇ ed to CR mRNAs, or to genomic CR sequences, can be used for both predi ⁇ ive and therapeutic evaluation of allelic mutations which might be manifest in a variety of disorders including cancer, immunodeficiencies, autoimmune disorders, developmental abnormalities, infe ⁇ ious diseases, toxic damage due to irradiation, chemicals, and other noxious compounds or natural products.
  • the nucleotide probes can help facilitate the determination of the molecular basis for a developmental disorder which may involve some abnormality associated with expression (or lack thereof) of a CR protein. For instance, variation in CR synthesis can be differentiated from a mutation in the CR coding sequence.
  • anti-sense techniques e.g., microinje ⁇ ion of antisense molecules, or transfe ⁇ ion with plasmids whose transcripts are anti-sense with regard to a CR mRNA or gene sequence
  • Such techniques can be utilized in cell culture, but can also be used in the creation of transgenic animals.
  • the present invention facilitates the development of assays which can be used to screen for drugs which are either agonists or antagonists of the normal cellular function of the subje ⁇ CR proteins, or of their role in cell signaling.
  • the invention in another aspe ⁇ , feamres transgenic non-human animals which express a recombinant CR gene of the present invention, or which have had one or more of the subject CR gene(s), e.g., heterozygous or homozygous, disrupted in at least one of the tissue or cell-types of the animal.
  • the invention features an animal model for disorders related to cell signaling, which has a CR allele which is mis-expressed.
  • Such a mouse model is suitable for smdying disorders arising from mis-expressed CR genes.
  • PBMCs Human peripheral blood mononuclear cells
  • T-cells were activated by stimulation of the CD3 component of the T-cell receptor complex with an anti-CD3 rea ⁇ ive monoclonal antibody (OKT3, 1:10,000 dilution, Ortho Pharmaceuticals, Raritan, NJ) in the presence of absence of 10 mg/ml CHX, and DNA synthesis was monitored at 48-52 hr by adding 0.5 mCi [ ⁇ H -thymidine to 200 ml aliquots of cell cultures in 96-well microtiter plates. Cultures were harvested onto glass fiber filters, radioa ⁇ ivity was counted by liquid scintillation, and [ ⁇ H]-_hymidine incorporation was calculated as cpm/10 4 cells/hr.
  • an anti-CD3 rea ⁇ ive monoclonal antibody OKT3, 1:10,000 dilution, Ortho Pharmaceuticals, Raritan, NJ
  • IL-2R-positive T-cell blasts were prepared by stimulation of PBMCs with OKT3 for 3 days, after which the cells were washed and replaced in culture for an additional 1 1 days in the presence of 500 pM IL-2. The cells were subsequently washed and placed in culture in the absence of IL-2 for 36 hr, followed by a 12 hr stimulation with 50 ng/ml phorbol 12,13 dibutyrate (PdBu) to augment high-affinity IL-2R expression. Cells were washed free of PdBU and placed in culture for 12 hr prior to restimulation. Such treatment enabled the generation of a GQ/GI -synchronized cell population, made up of greater than 90% T8-positive T lymphocytes (Gullberg et al., J. Exp. Med. 163: 270-284 (1986)).
  • IL-2R-positive T-cell blasts were cultured in the presence of 1 nM IL-2, 10 mg/ml CHX, 250 mM 4-thiouridine (Sigma Chemical Co., St. Louis, MO) and 2.5 mCi/ml [5,6- ]-uridine (48 Ci/mmole, Amersham, Arlington Heights, IL) for 2 hr.
  • CHX was included in the 2 hr IL-2 stimulation of the IL-2R-positive, GQ/GI -synchronized human T-cells from which the cDNA library was generated in order to isolate immediate-early genes, and also to possibly superinduce the expression of low-abundance messages.
  • Total RNA was isolated essentially as described by Caligiuri et al.
  • RNA Fra ⁇ ionation of total cellular RNA resulted in a 10-fold enrichment for newly-synthesized transcripts.
  • This thiol-sele ⁇ ed RNA was used in the synthesis of Not-1 primer/adapter-primed cDNA, utilizing the Riboclone cDNA Synthesis System (Promega, Madison, Wl) according to manufacturers instru ⁇ ions.
  • cDNA library was then screened using radiolabeled cDNA probes made from mRNA isolated from T-cells induced with IL-2 or from uninduced cells as follows.
  • Single-stranded [32p]_iabeled cDNA probes were prepared from poly(A) + RNA isolated from human T-cell blasts stimulated for 2 hr with medium (unstimulated probe), or 1 nM IL-2 and 10 mg/ml CHX (stimulated probe).
  • Total cellular RNA was prepared as described by Caligiuri et al. (Caliguri et al., J. Exp. Med.
  • RNA was isolated by three passages over an oligo-dT-cellulose column (5 Prime-3 Prime, West Chester, PA).
  • First strand cDNA synthesis was performed with an oligo-dT 12-18 primer (United States Biochemical Corp., Cleveland, OH), using the Riboclone cDNA Synthesis System (Promega, Madison, Wl) according to manufacturers instru ⁇ ions, with the exception of dCTP at a final concentration of 35 mM and the addition of 2.5 mCi/ml [ 3 P]-dCTP.
  • Hybridization was carried out for 72-96 hr at 42°C in 50% formamide, with a final probe concentration of approximately 2 x 10 ⁇ cpm/ml (W. M. Strauss, in Current Protocols in Molecular Biology, pp. 6.3.1-6.3.6 (1989)).
  • the initial screening yielded 18 putative positive clones which exhibited differential hybridization to the stimulated and unstimulated probes after three independent screens. These clones were isolated for further characterization by Northern Blot analysis.
  • Plasmids were purified from the clones of interest, and the Not-1- and EcoRI -excised inserts [32p]_iabeled with random primers. Hybridization was carried out in 50% formamide at 42°C for 48-72 hr, followed by repeated washes in 0.1 X SSC, 0.1 % SDS at 56-62°C (Selden, ibid.). Filters were exposed to Kodak XAR-5 film with Dupont Cronex intensifying screens, and specific bands quantitated with an EC densitometer (EC Apparatus Corp., St. Moscow, FL).
  • the original designations of the CR clones are included in parentheses in the left-hand column of Table X above.
  • the original designations are used herein to refer to the partial sequences shown in the column second from the left in Table II.
  • Table II and in Figures 8A-8H three of the genes, CRI, CR3, and CR5, were induced by IL-2 alone, while five of the genes, CR2, CR4, CR6, CR7, and CR8, were induced by both CHX and IL-2.
  • the combination of IL-2 and CHX resulted in a marked synergistic induction.
  • the temporal expression of the novel, IL-2-induced genes was determined by Northern blot analysis, using RNA isolated from human IL-2R-positive T-cell blasts after IL-2 stimulation in the presence or absence of CHX.
  • Northern blots were prepared with 15 mg total RNA isolated from GQ/GJ -synchronized human T-cells stimulated for 0, 0.5, 1, 2, 4, or 8 hours with 1 nM IL- 2 or IL-2 + 10 mg/ml CHX. Filters were probed with the cDNA inserts of the IL-2-induced clones.
  • IL-2-responsive genes have been found to range from those such as c-fos, which are rapidly and transiently induced within minutes of IL-2 stimulation (Dautry et al., J. Biol. Chem. 263: 17615-17620 (1988)), to those which remain at elevated levels through Gi to S phase entry (Sabath et al., J. Biol. Chem. 265: 12671-12678 (1990)).
  • the cDNA clones were subje ⁇ ed to sequence analysis.
  • Plasmids were isolated from the clones of interest essentially as described by Kraft et al., Biotechniques 6: 544-547 (1988)), and ve ⁇ or primers were used to sequence the termini of the cDNA inserts, employing the Sequenase 2.0 dideoxy sequencing kit (United States Biochemical, Cleveland, OH). Approximately 200 bases of sequence were attained from each end of the inserts. These partial sequences are described in Table II. Searches of the GenBank and EMBL data bases were performed with the FASTA program as described by Pearson et al., P.N.A.S. (USA) 85: 2444-2448 (1988).
  • the complete cDNA sequence (and the predi ⁇ ed amino acid sequence) of the eighth clone was determined to be identical to that of the IL-2 induced gene pim-1.
  • the nucleotide sequence as well as the predi ⁇ ed amino acid sequence of pim-1 are set forth at page 605 in Selten, G. et al., Cell 46: 603-611 (1986).
  • Northern blots were prepared as follows: Human IL-2R-positive T cells were treated with 1 nM IL-2 alone or in combination with 0.5 mM dibutyryl cAMP or sodium butyrate (NaBt) for 1, 2, or 4 hours. Filters were prepared with 15 mg total RNA and hybridized with cDNA inserts or the IL-2 induced clones.
  • RNA By isolation of RNA at early time intervals, it was possible to identify those genes which were induced by T-cell receptor triggering in the absence of IL-2 effects. Only one of the genes, 10D6, exhibited heightened levels of expression after 2 hr of T-cell receptor activation, while the seven others were apparently insensitive to this stimulus. Two of the clones, 1F5 and 11B2, were undetectable, even after seven days of autoradiographic exposure of the Northern blots. Two other genes, 11E6 and 13E2, were expressed at relatively high levels regardless of the stimulus; a ⁇ ivation with anti-CD3 did not induce RNA expression beyond the level observed by culture in medium alone. Identical results were obtained after 1 and 4 hr of stimulation.
  • IL-2 induced G progression genes Of the 8 IL-2 induced G progression genes reported here, only one appears to also be induced during the T cell receptor-mediated competence phase of the cell cycle. Thus, while several genes such as c-fos, c-myc and c-raf-1 are known to be induced during both the initial GQ- G ] and subsequent G S phase transitions, the expression of a number of IL-2-stimulated genes is unique to the latter event. In addition, the immediate-early genes reported here appear to define a class distin ⁇ from the IL-2-induced genes isolated by Sabath et al. (Sabath et al., J. Biol. Chem. 265: 12671-12678 (1990)).
  • Example VII Cloning and Analysis of CR8 As described above, the CR8 gene encodes a novel basic helix-loop-helix (bHLH) protein.
  • bHLH basic helix-loop-helix
  • CR8 transcript While the CR8 transcript is ubiquitously expressed in many tissues, it is induced by IL-2 as well as by IL-3 in cytokine-dependent lymphoid cell lines. In an IL-2-dependent human T cell line Kit 225, the CR8 transcript is induced not only by IL-2, but also by interferon b and forskolin, which elevates intracellular cAMP. The bHLH domain of CR8 shows the highest structural homology to a Drosophila transcriptional repressor hairy.
  • the recombinant CR8 protein binds preferentially to the Class B E-box DNA sequence (CACGTG), which is found in the promoter/enhancer regions of a number of genes associated with cell growth and differentiation, suggesting that CR8 may regulate the transcription of such genes.
  • CACGTG Class B E-box DNA sequence
  • CR8 contains a helix-loop-helix (HLH) domain, chara ⁇ eristic for transcription fa ⁇ ors.
  • the HLH domain is a dimerization motif chara ⁇ erized by the two amphipathic ⁇ -helices separated by a non-helical loop of variable length (Davis, R.L. et al., Cell 60: 733-746 (1990)).
  • HLH basic helix-loop-helix
  • HLH proteins A wide variety of developmental processes are regulated by HLH proteins; the MyoD family of myogenic transcription factors dire ⁇ ly induce the expression of muscle-specific genes, thereby fun ⁇ ioning as master regulators of muscle cell lineage specification (reviewed in (Edmonson, D. G. et al. (1993) J. Biol. Chem. 268: 755-758; Weintraub, H., Cell 75: 1241-1244 (1993)).
  • immunoglobulin (Ig) gene expression has been extensively studied, and has been shown to be controlled by numerous transcription fa ⁇ ors that recognize specific DNA sequences in the Ig enhancers (Kadesch, T., Immunol. Today 13: 31-36 (1992)).
  • Ig enhancers that recognize specific DNA sequences in the Ig enhancers.
  • E2A null mutant mice that lack mature B cells clearly depi ⁇ the impa ⁇ of these bHLH proteins on B cell development (Bain, G. et al., Cell 79: 885-892 (1994); Zhuang, Y. et al., Cell 79: 875-884 (1994)).
  • One of the most extensively smdied may be Myc, a bHLH protein encoded by the c-myc oncogene (reviewed in Marcu, K. B. et al., Annu. Rev. Biochem. 61: 809-860 (1992)).
  • the negative regulator Id proteins which inhibit differentiation by forming inactive heterodimers with bHLH proteins, thereby may be required for proliferation.
  • the level of Id expression is higher in undifferentiated proliferating cells (Benezra, R. et al. (1990) Cell 61: 49-59).
  • antisense oligonucleotide against Id mRNA inhibits re-entry to the cell cycle (Barone, M.V.
  • peripheral blood mononuclear cells were cultured in RPMI 1640 supplemented with 10% (v/v) heat-ina ⁇ ivated fetal calf serum (FCS) and antibiotics in the presence of OKT3 (Ortho Pharmaceuticals) for 3 days, then for an additional 11 days in the presence of IL-2 (Takeda Chemical).
  • FCS heat-ina ⁇ ivated fetal calf serum
  • OKT3 Ortho Pharmaceuticals
  • IL-2 Takeda Chemical
  • the cells were subsequently removed from of IL-2 for 36 hr, followed by a 12 hr stimulation with phorbol-12, 13-dibutyrate (Sigma) to augment the expression of high-affinity IL-2 receptor.
  • Kit 225 is an IL-2-dependent human T cell line (Hori, T. et al., Blood 70: 1069-1072 (1987)).
  • Ba/F3 and CTLL2 are mouse cell lines dependent on IL-3 and IL-2, respe ⁇ ively. Both Kit 225 and CTLL2 were maintained in RPMI 1640 supplemented with 10 % (v/v) FCS and 500 pM recombinant human IL-2.
  • Ba/F3 was maintained in RPMI 1640 supplemented with 10 % (v/v) FCS and 5 % (v/v) conditioned medium from fibroblasts transfe ⁇ ed with mouse IL-3 as a source of IL-3.
  • Recombinant mouse IL-3 was purchased from Genzyme. Before using for experiments, cell lines were made quiescent by growth fa ⁇ or deprivation for 72 hr for Kit 225, 12 hr for Ba/F3 and 2 hr for CTLL2. Forskolin was obtained from Sigma. Human interferon (IFN) ⁇ was from GIBCO BRL.
  • IFN Human interferon
  • Proliferation was monitored by measuring the incorporation of [3H-methyl]thymidine (Amersham) into ten thousand cells incubated with indicated reagents in 200 fl for 24 hr at 37 °C. The culture was pulsed with 0.5 fCi [ 3 H]thymidine for the last 4 hr prior to harvest.
  • RNAzolB Tel-Test
  • fra ⁇ ionated on a 1.2 % agarose formaldehyde gel RNA was visualized with ethidium bromide. After ele ⁇ rophoresis, RNA was transferred and fixed to Hybond-N+ nylon membrane (Amersham) with 40 mM NaOH. Multiple Tissue Northern Blot membranes were purchased from Clontech.
  • ⁇ gtlO cDNA libraries were constru ⁇ ed and screened according to the standard molecular biology procedure (Sambrook, J et al., Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)).
  • Poly(A)+ RNA was isolated from IL-2-stimulated normal human T cells prepared as above and cDNA synthesis was primed with both oligo(dT)12-18 and random hexamers.
  • the recombinant phages were screened with radiolabeled CR8 insert.
  • mouse CR8 a ⁇ gtlO cDNA library from IL-2-stimulated mouse splenocytes were screened wid human CR8 insert under low-stringency condition.
  • the recombinant CR8 protein with histidine-tag was prepared using the XpressTM System (Invitrogen) according to the manufacturer's protocol.
  • cDNA corresponding to the CR8 bHLH domain was obtained by PCR.
  • the PCR product was cloned into pT7Blue T-ve ⁇ or (Novagen) and subsequently into pRSET-A ve ⁇ or at BamHI and Hindlll sites. Constru ⁇ s were confirmed by DNA sequencing.
  • the protein was overexpressed in JM109 at 37°C in the presence of isopropylthio-b- D-gala ⁇ oside (IPTG) by infe ⁇ ing the ba ⁇ eria with M13 phages that contain the T7 RNA polymerase gene.
  • the cells were lysed witii 100 fg/ml lysozyme in native binding buffer (20 mM sodium phosphate, pH 7.8, 500 mM NaCl), the lysate was loaded on a ProBondTM Ni2+ column, and the recombinant protein was eluted with native-imidazole elution buffer (20 mM sodium phosphate, pH 6.0, 500 mM NaCl, 500 mM imidazole).
  • the protein was then dialyzed against lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 % glycerol, 0.1 % Triton X-100, 1 mM DTT) and concentrated on a Microcon 10 (Amicon). To confirm the purity and the quantity, the protein was fra ⁇ ionated on a 12% SDS-polyacrylamide gel with protein standards of known concentrations and visualized by silver staining. The recombinant protein corresponding to the bHLH domain of da was supplied by Dr. Michael Caudy (Cornell University Medical College).
  • the oligonucleotide probes used for the electrophoretic mobility shift assay are as follows: the Class A site used was the CACCTG hexamer (CAGGTG for the opposite strand) from the T5 promoter region of the Drosophila AS-C (Villares, R. et al., Cell 550: 415-424 (1987)) (5'-GATCGTAGTCACGCAGGTGGGATCCCTA-3' (SEQ. ID No.: 17) and 5'- GATCTAGGGATCCCACCTGCGTGACTAC-3' (SEQ.
  • the Class B site was the CACGTG hexamer from the USF binding site in the adenovirus major late promoter (Gregor, P. D. et al. (1990) Genes & Dev. 4:1730-1740) (5'- GATCGGTGTAGGCCACGTGACCGGGTGT-3' (SEQ. ID No.: 19) and 5'- GATCACACCCGGTCACGTGGCCTACACC-3') (SEQ. ID No.: 20), the Class C site was the CACGCG hexamer (CGCGTG for the opposite strand) from the AS-C T5 promoter (5'- GATCGGCAGCCGGCACGCGACAGGGCC-3' (SEQ.
  • oligonucleotide was labeled with [g- 32 P]ATP by T4 polynucleotide kinase, hybridized with three times excess of the opposite strand, and purified using MERmaid oligonucleotide purification kit (BIO 101).
  • 150 ng of the protein was allowed to bind to 50,000 cpm (equivalent to 0.5 ng in a typical experiment) of the labeled probe for 15 min at room temperature in 20 mM Hepes, pH 7.6, 50 mM KC1, 10 mM DTT, 5 % glycerol, 0.5 mM EDTA and 0.3 mg/ml BSA.
  • Two microgram of poly(dl-dC) was added to each 20 fl rea ⁇ ion as on-specific DNA. Samples were analyzed on a 5% native polyacrylamide gel and visualized by autoradiography.
  • CR genes were originally defined in IL-2 stimulated normal human T cells.
  • the level of CR8 expression was measured by Northern hybridization in the IL-2-dependent human T cell line Kit 225, the IL-3-dependent mouse pro-B cell line Ba/F3, and me IL-2-dependent mouse T cell line CTLL2.
  • a single 3.2 Kb species hybridized to the cDNA probe, and in all three cell lines tested, the level of CR8 was clearly augmented when the cells were stimulated with their respe ⁇ ive growth fa ⁇ ors. Correlation between die level of CR8 and that of DNA synthesis was in the presence of growth- inhibitory agents was also examined.
  • cytoplasmic cAMP increases in cytoplasmic cAMP are known to inhibit the growth of many cell types, including lymphocytes (Johnson, K. W. et al., P.N.A.S. (USA) 85: 6072-6076 (1988)). IFNs also exert antiproliferative a ⁇ ivity on many cell types (Pestka, S. et al., ) Annu. Rev. Biochem. 56: 727-777 (1987)). Therefore, Kit 225 was stimulated with IL-2, IFN ⁇ , or forskolin, which increases cytoplasmic cAMP by a ⁇ ivating adenylate cyclase, either alone or in combination.
  • IL-2-dependent [ 3 H]thymidine incorporation was inhibited by IFNb and forskolin in Kit 225 cells in a dose-dependent fashion.
  • IFN ⁇ and forskolin inhibit IL-2-dependent [ 3 H]thymidine incorporation by Kit 225 cells.
  • forskolin was capable of reducing the IL-2-dependent [ 3 H]thymidine incorporation almost to the basal level, IFN ⁇ - mediated inhibition never exceeded 70% of the maximal incorporation in several independent experiments.
  • the expression of CR8 was compared with that of c-myc, an IL-2-inducible immediate-early gene that encodes a bHLH protein and is implicated for cell proliferation (Marcu, K. B. et al. (1992) Annu.
  • CR8 transcripts were moderately induced, not only by IL-2, but also by IFN ⁇ or forskolin alone. Furthermore, the simultaneous stimulation of quiescent Kit 225 cells with IL-2 and IFN ⁇ , or IL-2 and forskolin, did not suppress the IL-2-induced expression of CR8 transcripts. In contrast, IL-2-indu ⁇ ion of c-myc expression was substantially inhibited in die presence of forskolin, while IFN ⁇ did not significantly reduce IL-2-promoted c-myc expression.
  • the original human CR8 clone isolated from the thiol-selected library had a 1.5 Kb insert, while the full-length mRNA transcript was estimated to be 3.2 Kb from Northern blotting experiments.
  • the CR8 clone did not have a long open reading frame, two full-length cDNA clones of human CR8 were isolated from a ⁇ gtlO human T cell cDNA library after two rounds of screening with cDNA fragments of the CR8 clone. These two clones were fully sequenced on both strands and the coding strand sequence determined (SEQ. ID No.: 13), and the amino acid sequence was deduced (SEQ. ID No.: 14).
  • a mouse CR8 cDNA fragment corresponding to nt 388 to 2720 of die human sequence was also isolated from a ⁇ gtlO mouse cDNA library by comparison of CR8 with other bHLH Proteins.
  • the protein database search with the putative peptide sequence revealed that CR8 shares homology with the bHLH proteins encoded by Drosophila hairy gene and the enhancer of split complex [E(spl)-C] of neurogenic genes.
  • Figures 1 A and IB show a sequence comparison of CR8 and other HLH proteins. Protein alignments were made to maximize homology within the bHLH domain. Amino acids conserved among most HLH proteins are shaded.
  • the proline residues in the basic region and the arginine residues at position 13 are boxed.
  • the boxed alanine residue in MyoD is the one whose substitution to proline abrogated the DNA binding and muscle-specific gene a ⁇ ivation a ⁇ ivity of MyoD (Davis, R. L. et al. (1990) Cell 60: 733-746). [h], human; [D], Drosophila melanogaster; [r], rat; and [m], mouse.
  • CR8 is a bHLH protein.
  • the amino acid sequence of the 58-residue bHLH domain of CR8 showed 40% identity to hairy, 41 % to E(spl)m7, and 45% to one of their mammalian counterparts HES-1. This degree of amino acid identity accounts well for the failure to detect any significant homology to any known bHLH proteins at the nucleotide sequence level.
  • the amino acid sequence for human and mouse CR8 was 100% identical in the bHLH domain.
  • Figure IB shows a sequence comparison of CR8 and hairy-related bHLH. conserveed amino acids are shaded. Note that HES-2, 3 and 5 proteins do not align perfectly in the hairy- related homology region (HRHR)-2.
  • HRHR hairy- related homology region
  • HHL human hairy-like
  • h(m) Drosophila melanogaster hairy [h(m)]
  • h(v) Drosophila virilis hairy [h(v)]
  • CR8 As shown in Figure 15B, the amino acid sequence of the bHLH region of CR8 is aligned with hairy, bHLH proteins of the E(spl)-C, deadpan (dpn) and their mammalian homologs (the term "hairy-related bHLH proteins" refer to them colle ⁇ ively).
  • hairy-related bHLH proteins refer to them colle ⁇ ively.
  • CR8 is the only one with a proline residue in the basic region, other than the hairy-related bHLH proteins.
  • the position of the proline residue is strictly conserved throughout the hairy-related bHLH proteins, in CR8 it is offset N-terminally by two residues.
  • CR8 and hairy-related bHLH proteins are different in the C-terminus as well; all the hairy-related bHLH proteins terminate with a specific Trp-Arg-Pro-Trp (WRPW) motif, which is absent in CR8. Nevertheless, CR8 showed appreciable homology to other hairy-related bHLH proteins in die region immediately C-terminal to me bHLH domain, which has been shown previously to be rich in hydrophobic residues, and proposed to form two more ⁇ -helices in bHLH proteins of the E(spl)-C (43). This region is referred to herein as the "hairy-related homology region (HRHR)-2", the HRHR-1 being the bHLH domain.
  • HRHR hairy-related homology region
  • the region N-terminal to the bHLH domain and the C-terminal half of the CR8 protein are rich in proline (8 proline residues between positions 1 and 30, 22 between 310 and 405). Notably, there are no known proteins in the data bases that share homologies to these most N-terminal and C-terminal regions of CR8.
  • CR8 Murre et al. (1989) Cell 58: 537-544, categorized bHLH proteins based upon tiieir tissue distribution. While proteins such as MyoD and AS-C gene produ ⁇ s show a cell-type specific expression, others such as E12/E47 and da are fairly ubiquitously expressed.
  • the tissue distribution of CR8 was analyzed using a Multiple Tissue Northern blot. CR8 transcripts of the expected size (3.2 Kb) were dete ⁇ ed in all tissues examined except placenta. This included heart, brain, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood leukocytes.
  • CR8 in peripheral blood leukocytes was unexpe ⁇ ed, in that CR8 is not expressed by quiescent T cells. This may reflect much higher sensitivity of Multiple Tissue Northern blot prepared from poly(A)+ RNA compared to our previous Northern blots, which used total RNA. Alternatively, the contribution of other leukocytes such as B cells, NK cells, monocytes and granulocytes that were not present in the original T cell preparations could account for CR8 expression by the peripheral blood leukocytes.
  • the canonical bHLH binding sequence is called d e E-box, CANNTG, originally identified in the immunoglobulin heavy chain enhancer (Ephrussi, A. et al., Science 227: 134-140 (1985)).
  • Many bHLH proteins were later divided into two mutually exclusive classes, depending on whether they bind to me Class A sites (CAGCTG/CACCTG) or the Class B sites (CACGTG/CATGTG) (Dang, C.V. et al., P.N.A.S. (USA) 89: 599-602 (1992)).
  • a histidine-tag was added to facilitate the purification of the recombinant protein. While most of the recombinant protein localized in inclusion bodies, there was still enough soluble protein in the cytoplasm, thereby enabling its purification under native conditions using a Ni 2 + column. A single band of protein was dete ⁇ ed at the expe ⁇ ed size (16.6 kD with the histidine-tag) by silver staining. EMSA was carried out using this recombinant protein.
  • CR8 bHLH protein strongly binds to the Class B (CACGTG) and the Class C (CACGCG) sites, and weakly to the N box (CACNAG) sequence but not to the Class A (CACCTG) site.
  • CACGTG Class B
  • CACGCG Class C
  • CACNAG N box
  • CACCTG Class A
  • the CR8 gene encodes a novel bHLH protein diat appears to fit into a class by itself.
  • CR8 is the first bHLH -containing protein found to be induced by cytokines.
  • CR8 clearly contains a bHLH motif most closely related to me hairy family, but the amino acid sequence of the basic region differs from other hairy-related proteins: the position of the proline residue is N-terminal to the defining proline of me hairy-related proteins, and CR8 lacks the C-terminal WRPW sequence found in all other hairy-related-related proteins.
  • CR8 binds preferentially to Class B sites.
  • IL-2 has been found to a ⁇ ivate the serine/threonine kinase proto-oncogene Raf-1 (Turner, B. et al., P.N.A.S. (USA) 88: 1227- 1231 (1991); Zmuidzinas, A. et al., Moi. Cell Biol. 11: 2794-2803 (1991)) and the tyrosine- specific kinases JAK 1 and JAK 3 (Beadling, C. et al., EMBO J. 13: 5605-5615 (1994); Miyazaki, T. et al., Science 266: 1045-1047 (1994); Russell, S.M. et al., Science 266: 1042-1045 (1994)).
  • Protein dimerization is more sele ⁇ ive than DNA binding, but currently no rules are available that predi ⁇ the dimerization preference of any given HLH proteins. Even so, a Class A-binding protein seems to form DNA binding heterodimers only with other Class A proteins, and a bHLH protein with a LZ does not form heterodimers with those wimout LZs (Blackwood, E. M. et al., Science 251: 1211-1217 (1991); Prendergast, G.C. et al., Cell 65: 395-407 (1991)). Therefore, if CR8 does form heterodimers, the most likely partner is a class B-binding bHLH protein wimout a LZ.
  • CR8 is most homologous to hairy in its bHLH domain, its preference for Class B E-box binding sites rather than class C sites, and its lack of a C-terminal WPRW motif, clearly sets CR8 apart and does not predi ⁇ necessarily that CR8 may a ⁇ as a transcriptional repressor as do hairy- related proteins.
  • Id proteins that lack a basic region have been shown to favor proliferation, presumably by forming heterodimers with differentiation inducing bHLH proteins, diereby preventing DNA binding and transcriptional a ⁇ ivation of genes that program differentiation (Barone, M. V. et al. (1994) Proc. Nad. Acad. Sci. USA 91: 4985-4988; Hara, E.
  • CR8 could promote proliferation by suppressing differentiation by either of these transcriptional repressor mechanisms.
  • CR8 could also a ⁇ ivate transcription like the bHLH-LZ Myc family.

Abstract

A method described produces a complementary DNA (cDNA) library enriched in ligand-inducible genes of a cell is described. Also described are novel cDNA libraries, specific ligand-induced nucleic acid sequences encoding ligand-induced proteins expressed by the cells, and proteins expressed by these DNAs, homologues, muteins, and fragments isolated using the method of the invention.

Description

LIGAND-STIMULATED GENE EXPRESSION BACKGROUND OF THE INVENTION
BACKGROUND OF THE INVENTION
Description of the Background
Mammalian cell growth, differentiation, and migration are directed by hormones and specific protein ligands, often termed cytokines. In particular, cells comprising the neuroendocrine, hematopoietic and the immune/inflammatory systems are known to be governed by cytokines. Cytokines, like other ligands, interact with cells by means of specific receptors, usually expressed on the cell surface.
A fundamental problem confronting biomedical scientists is to discern how signals are transduced through ligand receptors and how these signals determine the response of the cell. Many ligands influence their target cells by stimulating the expression of specific genes. However, the genes signaled by most cytokines remain largely unknown owing to the complexity of cellular biochemistry. Moreover, the gene products that are vital for performing different cellular processes are often only expressed transiently, and/or in very low concentrations so that they are difficult to detect, isolate and characterize.
Interleukin-2 (IL-2) is a cytokine that is critical for the immune system: it directs the proliferation and differentiation of T lymphocytes (T-cells), B lymphocytes (B-cells), and natural killer (NK) cells. Just how IL-2 signals these cellular events in the various types of target cells remains unknown. A few genes have been identified that are expressed as a result of IL-2 stimulation of T cells. These include the cellular protooncogenes c-fos, c-myb, c-myc, pim-1, and c-raf-1. However, exactly how many and what other genes are expressed as a result of IL- 2/IL-2 receptor interaction remains unknown. Since the discovery of DNA cloning, methods have become available to isolate specific genes expressed by cells. However, it has been difficult to devise new methods to isolate and clone all or most of the genes expressed by a cell activated by a given ligand, a task that must be done before one can understand how the ligand directs the cell to perform specific functions. In addition, methods of identifying a paπicular gene or genes stimulated early on after ligand receptor aαivation have not been easily forthcoming as the number of genes expressed constitutively is usually quite large, while those genes induced by the ligand are usually quite small, i.e., about 100 genes out of a total of about 10,000 genes.
Therefore, what is needed are methods to select and enrich only for those genes stimulated by a given ligand. Ideally, these methods should detect those genes expressed in low concentrations, as well as those expressed at high concentrations. SUMMARY OF THE INVENTION
This invention pertains to complementary deoxyribonucleic acid (cDNA) libraries enriched in clones containing genes induced by ligand stimulation of a cell having a corresponding receptor for the ligand, and to methods of producing the same. This invention also relates to the genes which are expressed immediately or early on as a consequence of such a ligand-receptor interaction, and to methods of identifying these genes. In accordance with the method of the invention, a cDNA library highly enriched in ligand-inducible genes is produced by activating a cellular receptor with a ligand to induce the expression of genes as a result of ligand-receptor binding, reverse transcribing these RNA, and differentially probing the cDNA and selecting clones that bind to induced cDNA, but not to uninduced cDNA. Useful ligands include any of those which can activate a specific cellular receptor, including natural or synthetic ligands for the receptor, e.g., cytokines such as the interleukins, cellular growth factors, colony stimulating factors, hormones, peptides, antibodies, and receptor-binding fragments thereof.
In one embodiment, the present invention relates to a cDNA library (ies) of nucleic acids induced by a specific ligand (s) and/or all redundant DNA sequences encoding the CR proteins, homologues and fragments, to a vector (s) carrying the library (ies), and to transfected cells carrying the hybrid vector (s) with the cloned library (ies). The invention also relates to specific cDNA sequences induced by interleukin-2 (IL-2) and all other DNA sequences encoding their expressed proteins, homologues and fragments, to the corresponding RNA sequences, to the polypeptides expressed by the sequences of the cDNA library, to analogues having about 60 to 99% homology with the nucleic acid and/or the polypeptide sequences thereof, and to fragments thereof about 1 to 200 amino acids long, and in some instances even longer.
A most preferred embodiment of the present invention relates to the Cytokine Response nucleic acids CRI, CR2, CR3, CR5, CR6, CR7, and CR8 identified below as SEQ. ID No: 1, 3, 5, 7, 9, 11, and 27, and all other nucleic acids encoding the same proteins, and to the respeαive proteins encoded by these nucleic acids identified below as SEQ. ID No: 2, 4, 6, 8, 10, 12, 14, or 28, and their glycosylated or deglycosylated analogues, homologues, and fragments. This invention also encompasses all DNA and RNA probes of smaller lengths encoding portions of the polypeptides of this invention. The genes and polypeptides of the invention may be utilized as diagnostic or therapeutic agents. The genes are suitable as targets for the development of assays to discover new drugs. The therapeutic agents are suitable for regulation of cellular proliferation and differentiation and, therefore, for the treatment of all kinds of cancers, immune diseases, such as allergic, autoimmune, and rheumatologic diseases, transplant rejeαion, and as anti-infeαives for fighting viral, baαerial, parasitic and fungal infeαions, among others. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
A cDNA library may be construαed, in accordance to this invention, which is enriched in nucleic acid sequences whose expression is induced by activation of a ligand-specific cellular receptor. This enriched library facilitates the identification and characterization of ligand-aαivated genes that are triggered immediately and/or early on after receptor activation, e.g., about 2 to 4 hours after the ligand binds to its receptor. Such genes are believed to play a role in stimulating growth phase transitions and subsequent clonal expansion of a particular cell type.
The cells are aαivated by a ligand in the presence of labeled RNA precursors, which are incorporated into the new RNA synthesized by the cell in response to receptor activation. Labeled precursors are used in order to distinguish newly transcribed from unlabeled, preexisting RNA. Preferred labeled RNA precursors include 6-thioguanine, 4-thiouridine, and tritiated uridine, but others may also be utilized. Aαivation is also carried out in the presence of a substance which enhances the level of RNA in a cell. Preferred substances include protein synthesis inhibitors, such as cycloheximide and puromycin, although others may be used, such as cyclic 3',5'-adenosine monophosphate (cAMP), analogs of cAMP such as dibutyryl cAMP, and other molecules which increase the intracellular level of cAMP. The labeled RNA is then separated from the unlabeled RNA and used to prepare cDNA. The cDNA is cloned into a rector to provide a library of cDNA-containing clones. This library is then screened for clones containing ligand-inducible genes. In one embodiment, the screening step includes probing the cDNA library with a DNA probe constructed from total cellular RNA or mRNA derived from (1) a ligand-induced cell and (2) an uninduced cell. The library is probed under conditions such that the probe hybridizes specifically with a complementary cDNA sequence in the library. The seleαing step includes seleαing those clones containing sequences that hybridize only with probes constructed from ligand-induced mRNA or total RNA. By following the method of the invention, and inducing T-lymphocytes with IL-2, eight clones containing ligand-induced genes have been identified. These genes have been named Cytokine Response (CR) genes 1-8, of which CR 1-3, 5, 6, and 8 are novel. CR4 is identical to a gene reported as SATB-1 (Dickinson, L. A. et al. (1992) Cell 70:631-645), for Special AT-rich Binding protein 1, which binds seleαively to the nuclear matrix/scaffold- associating region of DNA. CR7, also identified using the method of the invention, is identical to the putative proto-oncogene, pirn 1 , a known IL-2-induced gene. The nucleic acid sequences of these CR genes, i.e., CR genes 1-8 are set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 and 27. The amino acid sequences encoded by these CR genes are set forth in SEQ ID NOs.: 2, 4, 6, 8, 10. 12. 14. and 28. The method of the invention may be used to create cDNA libraries of the genes induced by aαivation of a variety of different cellular receptors. The receptors may be cytoplasmic, nuclear, or cell-surface receptors, and include receptors for cytokines, hormones, faαors, and peptides, among other types of receptors. For example, cytokines such as the interleukins (e.g., IL-1 and IL-2), cellular growth faαors (e.g., platelet-derived growth faαor (PDGF), epidermal growth faαor (EGF), fibroblast growth faαor (FGF), insulin-like growth faαor (IGF)), colony stimulating faαors (e.g., multiplication stimulating aαivity), and hormones (e.g., insulin, somatomedin C, and steroid hormones) are useful as aαivators of certain cellular receptors. The ligand used to activate the receptor may be the natural ligand recognized by the receptor or a synthetic analogue or fragment. Alternatively, an antibody specific for the receptor and capable of aαivating the receptor may also be used. The receptor is, thus, aαivated by a ligand or other means of aαivation for a predetermined length of time and at an effeαive concentration. This activation may be carried out in the presence of labeled RNA precursors which are incorporated into the RNA synthesized by the cell in response to receptor aαivation. Thus, the RNA transcribed is labeled so as to be distinguished from preexisting RNA which is not labeled. Some labels (such as radiolabels) may be employed to monitor the newly synthesized RNA. Useful radiolabeled RNA precursors for such purposes include [^H]-uridine. Other labels may be used to separate newly transcribed RNA from unlabeled RNA. For example, RNA synthesized from thiol-labeled RNA precursors specifically adheres to phenylmercury agarose (Woodford et al., Anal. Biochem. 1781: 166-172 (1988)). RNA newly synthesized in response to receptor aαivation may be separated from preexisting RNA in the cells. All RNA molecules expressed prior to ligand-aαivation pass through the phenylmethyl mercury column, leaving only the newly synthesized, thiol- (SH-) labeled RNA attached to the agarose via a covalent bond between the mercury and sulfur. The thiolabeled RNA molecules may then be eluted from the column by reducing the Hg-S bond with an excess of 2-mercaptoethanol. To augment the expression of immediate/early ligand-activated genes, which may be difficult to identify because of the large number of constitutively expressed genes, a substance that enhances the level of RNA may be added to the culture medium during the ligand stimulation (see, e.g., Cochran et al., Cell 33: 939-947 (1983)). Useful substances include those compounds that stabilize RNA and/or that block translation, thereby blocking feedback inhibition of these genes by a later gene product. Such aαivity may potentiate the magnitude of the RNA expressed as well as the duration of the life of the RNA. Examples of such useful substances include cycloheximide (CHX), which inhibits protein synthesis at the level of RNA-ribosome complexing and may stabilize polysomal RNA, and puromycin, which inhibits translation by causing premature dissociation of the peptide- mRNA-ribosome complex. The labeled RNA transcribed consequent to receptor aαivation in the presence of the substance which enhances RNA levels may then be purified from the cytoplasm of the cells. The purification may be accomplished by extraαing total cellular RNA from a cell homogenate or fraction thereof, isolating mRNA therefrom, for example, using a poly U or poly [dT] column, and then separating the labeled RNA from the unlabeled RNA. The separation may be accomplished, for example, using the phenylmethyl mercury agarose protocol described above. Of course, other known methods of separating the newly synthesized RNA from the preexisting can also be used.
A cDNA library (ies) may be prepared from the separated labeled RNA by standard techniques. For example, the labeled RNA may be reversed transcribed into cDNA, using oligo [dT] primers. The cDNA may then be ligated into appropriate veαors using established recombinant DNA techniques. A cDNA library is then construαed by methods well known in the art and transfected into prokaryotic or eukaryotic host cells. Prokaryotic systems most commonly utilize E. coli as host, although other baαerial strains such as Bacillus, Pseudomonas, or other Gram-positive or Gram-negative prokaryotes may also be used. When such prokaryotic hosts are employed, operable control systems compatible with these hosts are ligated to the cDNA fragments and disposed on a suitable transfer veαor which is capable of replication in the baαerial host cell. Backbone vectors capable of replication include phage and plasmid veαors. Common plasmid veαors include those derived from pBR322 and the pUC series. One such useful vector which is commercially available is the plasmid p Bluescript ΗISK+ (Stratagene, La Jolla, CA). Charon lambda phage is a frequently employed phage vector. Others, however, may also be employed. Control sequences obligatorily include promoter and ribosome binding site encoding sequences, and a variety of such controls are available, in the an, such as the beta- laαamase (pencillinase) and laαose (lac) promoter systems (see, e.g., Chang et al., Nature 198: 106 (1977)), and the tryptophan (trp) promoter systems (Goeddel et al., Nucleic Acids Res. 8: 4057 (1980)). Composite promoters containing elements of both the trp and lac promoter systems are also available in the art. Eukaryotic microbes, such as laboratory strains of Saccharomyces cerevisiae, or Baker's yeast, can also be used for expression. A number of yeast control systems and veαors are available, including those which are promoters for the synthesis of glycolytic enzymes (see, e.g., Hess et al., Biochem. 17: 4900 (1968)). Yeast vectors, e.g., employing the 2 micron origin of replication are suitable as transfer veαors (see, e.g., Broach, Meth. Enzym. 101: 307 (1982)). Tissue cultures of insect cell lines, or cell lines immortalized from mammalian or other higher organisms have also been used as recombinant hosts. Such cell lines include Chinese hamster ovary (CHO), Vero, HeLa, and COS cells. In general, the COS cell system is used for transient expression, while CHO cells typically integrate transformed DNA into the chromosome. Suitable mammalian veαors are generally based on viral origins of replication and control sequences. Most commonly used are the simian virus 40 (SV40) promoters and replicons (See, Fiers et al., Nature 273: 113 (1978)) and similar systems derived from Adenovirus 2, bovine papilloma virus, and avian sarcoma virus.
The ligand-activated genes are then screened in the library using any one of several different methods. One method involves differential hybridization with cDNA probes constructed from mRNA derived from ligand-activated cells and unaαivated cells. Another method includes hybridization subtraction, whereby cDNA from ligand-aαivated cells is hybridized with an excess of mRNA from unactivated cells to remove RNA molecules common to both. Alternatively, cDNA probes may be made from the same pool of thiol-seleαed mRNA used to make the cDNA library, as these sequences are highly enriched for ligand-induced molecules. cDNA probes may be prepared from mRNA. extracted from cells treated with drugs that block the biological response to the particular cytokine (e.g., rapamycin and elevated cAMP block the proliferative response of T cells to IL-2, and cyclosporin A and FK506 block the T-cell response to activation via the T- cell antigen receptor). Results from probing with the cDNA made from drug-inhibited cells may then be compared to results from probes made from cells not inhibited by these drugs.
The marked superinduction observed for a number of the genes using a substance, such as CHX, which enhances RNA levels, is crucial in enabling their detection by differential hybridization, as it has been estimated that differential hybridization is only effeαive in the detection of relatively high-abundance RNAs expressed at a level of greater than about 500 copies per cell. Therefore, the superinduction increases that level of expression of low-abundance RNAs above the threshold of detection by differential screening. In addition, the about 10-fold enrichment for newly synthesized RNA afforded by the thiol-labeling procedure further heightens the efficacy of the cloning procedure. Thus, the combination of superinduαion and thiol-labeling of RNA significantly enhances the sensitivity of differential screening, and provides a cloning strategy which has the capacity to deteα messages normally present in relatively low abundance (i.e., less than about 100 copies/cell). After the initial screening of the cDNA library, all clones isolated as tentatively positive must be corroborated as truly ligand-aαivated. This can be accomplished by isolating the cDNA insert from each cloned plasmid, and then employing this cDNA to probe RNA from ligand-aαivated cells by Northern blot analysis. Then, to identify each gene, the cDNA may be subjected to sequence analysis. Searches of the GenBank (Los Alamos, NM) and EMBL (Heidleberg, Germany) data bases can be made of even partial sequences to identify known sequences such as pim-1, a previously charaαerized, IL-2 induced gene.
A number of methods may be used to charaαerize the novel ligand-enhanced genes and begin to determine their functional roles in, for example, signal transduαion. An analysis of the cDNA corresponding to the mRNA transcript can be used to prediα the coding region for the gene product and its amino acid sequence. From the amino acid sequence, the gene produα can be placed into one of several categories of proteins, such as DNA-binding proteins, kinases, phosphatases, transmembrane proteins, or secreted products. The analysis of the regulatory elements governing expression of the immediate-early genes described in the present study is useful in the charaαerization of the secondary biochemical messengers activated by the ligand, e.g., IL-2 receptor. Other methods helpful in determining the functional relevance of the IL-2- induced genes include examining T-cells for their expression in response to triggering of other receptors.
One such receptor is the T-cell antigen receptor. Seminal studies of the T-cell system have demonstrated that T-cell aαivation occurs as a two-step process. Quiescent cells are initially stimulated through engagement of the antigen receptor, which provides the cells with the capacity to produce and respond to IL-2. Subsequently, the interaction of IL-2 with its cell-surface receptor drives progression through the Gj to the S phase of the cell cycle. Transmembrane signaling through both the T-cell antigen receptor has been shown to trigger the heightened expression of a number of genes, including c-fos, c-myc and c-raf-1 (Reed et al., P.N.A.S. (USA) 83: 3982-3986 (1986); Dautry et al., J. Biol. Chem. 263: 17615-17620 (1988); Zmuidzinas et al., Moi. Cell. Biol. 11: 2794-2803 (1991)). By comparison, in the case of the c-myb gene, the induαion is unique to the IL-2 signaling pathway (Stern et al., Science 233: 203-206 (1986)). Therefore, to categorize the novel IL-2-induced genes with regard to their patterns of induαion by these two receptor pathways, the sensitivity of the genes to T-cell receptor stimulation can be determined. Additional methods that can be used to categorize the genes isolated include screening for expression by proliferating versus non-proliferating cells, for tissue-specific expression, and for expression in response to different cytokines and hormones. Genes that are expressed exclusively by proliferating cells, very likely funαion to promote cell growth. Other genes signal differentiation and would be expected to be tissue-specific or aαivated only by a restriαed family of similar ligands. An additional means of establishing the mechanisms of IL-2-mediated transmembrane signaling is provided by the varied effeαs of elevated cAMP on IL-2-induced gene expression. The diverse responses of the genes to cAMP suggest that the IL-2 signaling pathways responsible for their induαion bifurcate at a point prior to intersection with the cAMP regulated pathways. One potential mechanism of cAMP aαion very likely involves regulation of protein phosphorylation, as cAMP is an activator of protein kinase A, and elevations of intracellular cAMP inhibit IL-2-inducted phosphorylation events. In addition, as cAMP blocks IL-2-stimulated cell cycle progression at a point in early G\, cAMP sensitivity is a useful tool with which to dissect IL-2-mediated signal transduαion pathways involved in cell cycle progression. A likely funαion of the immediate/early gene produαs is the governing of subsequent DNA replication and cell division. Previously characterized IL-2 induced genes encode kinases (c- raf-1, pim-1) and DNA binding proteins (c-fos, c-myc, c-myb). Sequence analysis of the novel genes then determines whether the proteins they encode contain conserved domains which would implicate similar funαions. However, IL-2 stimulates cellular differentiation as well as division, and has been shown to induce the expression of a number of genes to perform roles other than in cell cycle progression. cAMP sensitivity indireαly establishes involvement in G\ progression. The demonstration of induαion of the genes by other growth factors, as well as heightened expression in transformed cell lines further supports this notion. A more direct approach, utilizing antisense oligonucleotides, helps to determine whether specific blockage of expression of any of these genes is sufficient to prevent cell cycle progression. Similarly, the immediate early gene products may exert cell cycle control through the induction of expression of late genes, as has been demonstrated for regulation of the PCNA/cyclin, DNA polymerase A and cdc2 genes by the c-myb and c-myc gene products. Interestingly, the IL-2-induced expression of the PCNA/cyclin and DNA topoisomerase II gene in late G is specifically inhibited by cAMP, so that cAMP sensitivity of immediate early gene expression provides a test for the genes that play a role in regulating late gene expression. If, like the previously characterized cell cycle regulatory cdc2/CDC28 and cyclin genes, the novel IL-2 induced genes are highly conserved, then it is possible to isolate yeast homologs of the clones and perform deletional analyses to further define the funαions of the gene produαs. The definitive assignment of a funαion to a given gene product depends upon a series of different approaches, including determining intracellular location, and determining the consequences of blocking the expression of the gene either by mRNA antisense methods or by homologous recombination methods. All of the methods necessary for these studies exist as prior art and therefore, given the identification of a given gene as aαivated by a ligand such as the cytokine IL-2, the characterization of the product is attained.
By following the method of the invention, the inventors have identified eight clones containing IL-2-induced genes. At least six of these ligand-induced genes are novel and have been named Cytokine Response (CR) genes 1-3, 5, 6 and 8. Of the remaining two, CR4 is identical to a gene reported as SATB-1 (Dickinson, L. A. et al., Cell 70: 631-645 (1992)), for Special AT- rich Binding protein 1, which binds selectively to the nuclear matrix/scaffold-associating region of DNA, and CR7 is identical to the putative proto-oncogene, pirn 1, a known IL-2-induced gene. Table I below provides several characteristics of the CR genes. Table I: CR Gene Characteristics
Figure imgf000011_0001
The nucleic acid sequences of these CR genes, i.e., CR genes 1 to 8, are set forth in Tables II to VIII below, as SEQ. ID Nos: 1, 3, 5, 7, 9, 11, 13, and 27, or fragments thereof. The amino acid sequences encoded by these CR genes are also provided below as SEQ. ID Nos: 2, 4, 6, 8, 10, 12, 14, and 28.
The regulatory regions of all of the CR genes may be used to construα assays to identify the relevant cis-aαing DNA response elements, the trans-aαing faαors responsible for transcriptional activation leading to CR gene expression, and the biochemical signaling for pathways triggered by IL-2 (the ligand) that aαivate the transcriptional aαivating faαors. The regulatory regions of all of the CR genes may also be used to construα assays useful for drug discovery. Thus, the promoter regions of the CR genes, which are 5' to the coding regions identified from the cDNA sequences, are used to construα a promoter-reporter assay to analyze signal transduαion pathways. Fragments of DNA derived from genomic DNA are engineered upstream from "reporters", such as luciferase. These promoter-reporter construαs are transfeαed into cells that are ligand-inducible, and then used as rapid and convenient assays for ligand- induced gene activation. Such assay is used in drug-discovery screening systems, where natural produαs or synthetic chemicals are tested. Those agents showing high negative or positive aαivity as regulators of the CR gene promoter-reporter assays, are suitable as therapeutic agents, and their chemical struαures may be modified by traditional medicinal chemistry, to provide congeners with further enhanced activities. The agents exhibiting aαivity in the CR gene promoter-repoπer assays are then tested in vivo, in cellular proliferation/differentiation systems, and/or in animals and humans, for anti-proliferative aαivity. These assays can be used to identify novel agents or drugs that either suppress or aαivate CR gene expression. Such novel agents or drugs may be used as immunosuppressives, immunosiimulants, antiinfeαives, or anti-cancer agents. The immediate-early CR genes and gene produαs may also be used to construct assays to determine the biochemical and molecular events, initiated by the ligand-receptor stimulation, that promote progression to the intermediate and late stages of the cell cycle responsible for DNA synthesis and replication. These assays are also used to identify novel agents and drugs that either suppress or promote these processes. With the capacity to generate large quantities of the CR gene products, the three-dimensional structures of the products are determined by conventional methods, such as x-ray crystallography and nuclear magnetic resonance. From this information, novel agents or drugs are identified, e.g., using computer analysis of the chemical structures, that interact with the CR gene product. These agents may be used as therapeutic agents.
CRI expression is rapidly and transiently induced by IL-2, and mRNA expression is suppressed by elevated intracellular cAMP, which also suppresses IL-2-promoted G\ progression. There are available pharmaceuticals that elevate intracellular cAMP, such as aminophylline and theophylline. Therefore, it is now possible to determine how these agents ftmαion to inhibit CRI expression and to identify novel agents that act similarly, but may have particular pharmacologic advantages. The CRI gene includes 2406 nucleotides of the coding fragment of SEQ. ID NO: 1 encoding a 202 amino acid protein (about 22 RDA) of SEQ. ID NO: 2, that shares sequence homology to two other recently reported genes, GOS8 and BL-34, both of which are induced to high levels of expression by mitogens. The nucleotide sequence of the CRI gene is about 58% homologous to the nucleotide sequence of the GOS8 gene (Siderovski, D. P., et al., DNA and Cell Biology 13: 125-147 (1994)), which was isolated from a PHA-induced T cell library. At the protein level, CRI is about 51.2% homologous to GOS8. In addition, the nucleotide sequence of the CRI gene is about 58% homologous to the nucleotide sequence of the BL34 gene (Hong, J.X. et al., J. Immune. 150: 3895-3904 (1993)), which was isolated from a Staph A-activated B cell cDNA library. Recently, 13 new homologues of CRI were reported (see, Druey, K.M. et al. Nature 379: 742-746 (1996)). These genes and gene produαs are an entirely new molecular family, that has been named the RGS family, for Regulators of G-protein Signaling. These CRI homologues, including BL34 (RGS-1) and GoS8 (RGS-2) have been shown to inhibit signaling via G-proteins, which typically couple to a class of receptors that have 7 membrane spanning units. The RGS proteins are said to interact with the alpha subunit of the trimeric G-protein complex. In the CRI case, elevated intracellular cAMP completely suppresses mRNA expression and G-protein-coupled receptors often elevate cAMP. This highlights an important correlation between IL-2-induced CRI and cAMP. At the protein level, CRI is about 48.0% homologous to BL34. The homology of CRI with BL-34 is of particular interest, in that BL-34 is expressed only by activated B cells, is preferentially expressed in vivo by B cells in lymph node germinal centers, and is overexpressed in B cell malignancies. As prediαed from its amino acid sequence which contains neither a hydrophobic leader sequence nor a transmembrane region, CRI is an intracellular protein. Also, the CRI protein includes no sequences consistent with other functional motifs or domains, such as found for DNA binding proteins, kinases, phosphatases, or linker molecules. The sequenced DNA and deduced protein sequence for CRI are provided below in Table II.
Table II: Full Sequenced DNA and Deduced Protein Sequence for CRI
AACCCAACCG CAGTTGACTA GCACCTGCTA CCGCGCCTTT GCTTCCTGGC GCACGCGGAG 60 CCTCCTGGAG CCTGCCACCA TCCTGCCTAC TACGTGCTGC CCTGCGCCCG CAGCC ATG 118
Met 1 TGC CGC ACC CTG GCC GCC TTC CCC ACC ACC TGC CTG GAG AGA GCC AAA 166 Cys Arg Thr Leu Ala Ala Phe Pro Thr Thr Cys Leu Glu Arg Ala Lys
5 10 15
GAG TTC AAG ACA CGT CTG GGG ATC TTT CTT CAC AAA TCA GAG CTG GGC 214 Glu Phe Lys Thr Arg Leu Gly lie Phe Leu His Lys Ser Glu Leu Gly 20 25 30
TGC GAT ACT GGG AGT ACT GGC AAG TTC GAG TGG GGC AGT AAA CAC AGC 262 Cys Asp Thr Gly Ser Thr Gly Lys Phe Glu Trp Gly Ser Lys His Ser
35 40 45
AAA GAG AAT AGA AAC TTC TCA GAA GAT GTG CTG GGG TGG AGA GAG TCG 310 Lys Glu Asn Arg Asn Phe Ser Glu Asp Val Leu Gly Trp Arg Glu Ser 50 55 60 65
TTC GAC CTG CTG CTG AGC AGT AAA AAT GGA GTG GCT GCC TTC CAC GCT 358 Phe Asp Leu Leu Leu Ser Ser Lys Asn Gly Val Ala Ala Phe His Ala 70 75 80 TTC CTG AAG ACA GAG TTC AGT GAG GAG AAC CTG GAG TTC TGG CTG GCC 406 Phe Leu Lys Thr Glu Phe Ser Glu Glu Asn Leu Glu Phe Trp Leu Ala
85 90 95
TGT GAG GAG TTC AAG AAG ATC CGA TCA GCT ACC AAG CTG GCC TCC AGG 454 Cys Glu Glu Phe Lys Lys lie Arg Ser Ala Thr Lys Leu Ala Ser Arg 100 105 110
GCA CAC CAG ATC TTT GAG GAG TTC ATT TGC AGT GAG GCC CCT AAA GAG 502 Ala His Gin lie Phe Glu Glu Phe lie Cys Ser Glu Ala Pro Lys Glu
115 120 125
GTC AAC ATT GAC CAT GAG ACC CGC GAG CTG ACG AGG ATG AAC CTG CAG 550 Val Asn lie Asp His Glu Thr Arg Glu Leu Thr Arg Met Asn Leu Gin 130 135 140 145
ACT GCC ACA GCC ACA TGC TTT GAT GCG GCT CAG GGG AAG ACA CGT ACC 598 Thr Ala Thr Ala Thr Cys Phe Asp Ala Ala Gin Gly Lys Thr Arg Thr 150 155 160 CTG ATG GAG AAG GAC TCC TAC CCA CGC TTC CTG AAG TCG CCT GCT TAC 646 Leu Met Glu Lys Asp Ser Tyr Pro Arg Phe Leu Lys Ser Pro Ala Tyr
165 170 175
CGG GAC CTG GCT GCC CAA GCC TCA GCC GCC TCT GCC ACT CTG TCC AGC 694 Arg Asp Leu Ala Ala Gin Ala Ser Ala Ala Ser Ala Thr Leu Ser Ser 180 185 190
TGC AGC CTG GAC CAG CCC TCA CAC ACC T GAGTCTCCAC GGCAGTGAGG 742
Cys Ser Leu Asp Gin Pro Ser His Thr (SEQ. ID No: 2)
195 200
AAGCCAGCCG GGAAGAGAGG TTGAGTCACC CATCCCCGAG GTGGCTGCCC CTGTGTGGGA 802 GGCAGGTTCT GCAAAGCAAG TGCAAGAGGA CAAAAAAAAA AAAAAAAAAA AAAAATGCGC 862 TCCAGCAGCC TGTTTGGGAA GCAGCAGTCT CTCCTTCAGA TACTGTGGGA CTCATGCTGG 922 Table II: Full Sequenced DNA and Deduced Protein Sequence for CRI (Cont'd)
AGAGGAGCCG CCCACTTCCA GGACCTGTGA ATAAGGGCTA ATGATGAGGG TTGGTGGGGC 982
TCTCTGTGGG GCAAAAAGGT GGTATGGGGG TTAGCACTGG CTCTCGTTCT CACCGGAGAA 1042 GGAAGTGTTC TAGTGTGGTT TAGGAAACAT GTGGATAAAG GGAACCATGA AAATGAGAGG 1102
AGGAAAGACA TCCAGATCAG CTGTTTTGCC TGTTGCTCAG TTGACTCTGA TTGCATCCTG 1162
TTTTCCTAAT TCCCAGACTG TTCTGGGCAC GGAAGGGACC CTGGATGTGG AGTCTTCCCC 1222
TTTGGCCCTC CTCACTGGCC TCTGGGCTAG CCCAGAGTCC CTTAGCTTGT ACCTCGTAAC 1282
ACTCCTGTGT GTCTGTCCAG CCTTGCAGTC ATGTCAAGGC CAGCAAGCTG ATGTGACTCT 1342 TGCCCCATGC GAGATATTTA TACCTCAAAC ACTGGCCTGT GAGCCCTTTC CAAGTCAGTG 1402
GAGAGCCCTG AAAGGAGCCT CACTTGAATC CAGCTCAGTG CTCTGGGTGG CCCCCTGCAG 1462
GTGCCCCCTG ACCCTGCGTT GCAGCAGGGT CCACCTGTGA GCAGGCCCGC CCTGGGCCCT 1522
CTTCCTGGAT GTGCCCTCTC TGAGTTCTGT GCTGTCTCTT GGAGGCAGGG CCCAGGAGAA 1582
CAAAGTGTGG AGGCCTCGGG GAGTGACTTT TCCAGCTCTC ATGCCCCGCA GTGTGGAACA 1642 AGGCAGAAAA GGATCCTAGG AAATAAGTCT CTTGGCGGTC CCTGAGAGTC CTGCTGAAAT 1702
CCAGCCAGTG TTTTTTGTGG TATGAGAACA GCCAAAAAGA GATGCCCCGA GATAGAAGGG 1762
GAGCCTTGTG TTTCTTTCCT GCAGACGTGA GATGAACACT GGAGTGGGCA GAGGTGGCCC 1822
AGGACCATGA CACCCTTAGA GTGCAGAAGC TGGGGGGAGA GGCTGCTTCG AAGGGCAGGA 1882
CTGGGGATAA TCAGAACCTG CCTGTCACCT CAGGGCATCA CTGAACAAAC ATTTCCTGAT 1942 GGGAACTCCT GCGGCAGAGC CCAGGCTGGG GAAGTGAACT ACCCAGGGCA GCCCCTTTGT 2002
GGCCCAGGAT AATCAACACT GTTCTCTCTG TACCATGAGC TCCTCCAGGA GATTATTTAA 2062
GTGTATTGTA TCATTGGTTT TCTGTGATTG TCATAACATT GTTTTTGTTA CTGTTGGTGC 2122
TGTTGTTATT TATTATTGTA ATTTCAGTTT GCCTCTACTG GAGAATCTCA GCAGGGGTTT 2182
CAGCCTGACT GTCTCCCTTT CTCTACCAGA CTCTACCTCT GAATGTGCTG GGAACCTCTT 2242 GGAGCCTGTC AGGAACTCCT CACTGTTTAA ATATTTAGGT ATTGTGACAA ATGGAGCTGG 2302
TTTCCTAGAA ATGAATGATG TTTGCAATCC CCATTTTCCT GTTTCAGCAT GTTATATTCT 2362
TATGAAATAA AAGCCCAAGT CCAATATGAA AAAAAAAAAA AAAA (SEQ. ID No: 1) 2406
The present invention, therefore, also pertains to a CRI polypeptide, preferably a substantially pure preparation of a CRI polypeptide, or a recombinant CRI polypeptide. In preferred embodiments, the CRI polypeptide comprises an amino acid sequence at least about 60 to 95%, preferably about 75 to 85% homologous to the amino acid sequence in SEQ ID NO: 2, the polypeptide has an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NO: 2, the polypeptide comprises at least about 5 to 200, preferably about 10 to 150, more preferably about 20 to 100 amino acids in length. The polypeptide comprises at least about 5, preferably at least about 10, more preferably at least about 20, more preferably at least about 50, 100, or 150 contiguous amino acids from SEQ ID NO: 2. In further preferred embodiments, a protein homologous to SEQ ID NO: 2 has a molecular weight of about 15-30 kD, preferably about 22 kilodaltons (kD). In a preferred embodiment, a polypeptide having at least one biological activity of the CRI polypeptide may differ in amino acid sequence from the sequence in SEQ ID NO: 2, but such differences result in a modified polypeptide which funαions in the same or similar manner as or which has the same or similar characteristics of native CRI protein. Such a peptide can include at least about 1 to 30, preferably about 2 to 20, and more preferably 10 to 15 amino acid residues from residues 1-202 of SEQ ID NO: 2. In yet other preferred embodiments, the CRI polypeptide is a recombinant fusion protein which includes a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated to the SEQ ID NO: 2, e.g., the second polypeptide portion comprises glutathione-S-transferase, a DNA binding domain, a polymerase activating domain so that, e.g., the fusion protein is funαional in a two-hybrid assay. Yet another aspect of the present invention concerns an immunogen comprising a CRI polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the CRI polypeptide; e.g., a humoral response, an antibody response, a cellular response. In preferred embodiments, the immunogen comprises an antigenic determinant, e.g., a unique determinant, from a protein comprising SEQ ID NO:2 analogues or fragments thereof . A further aspect of the present invention features a monoclonal or polyclonal antibody specifically reaαive with an epitope of the CRI immunogen, which is prepared as is known in the art.
Another aspect of the present invention provides a substantially pure nucleic acid comprising a nucleotide sequence which encodes a CRI polypeptide, analogues and/or fragments thereof. In certain preferred embodiments, the subjeα CRI nucleic acid includes a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the CRI gene sequence, e.g., to render the CRI gene sequence suitable for use as an expression veαor. In yet a further preferred embodiment, the nucleic acid which encodes a CRI polypeptide of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least about 12 to 300, more preferably to at least about 20 to 250, and more preferably to at least about 40 consecutive nucleotides of SEQ ID NO: 1. In yet a further preferred embodiment, the CRI encoding nucleic acid hybridizes to a nucleic acid probe corresponding to a subsequence encoding at least about 4 to 200, preferably at least about 10 to 150, and even more preferably at least about 20 to 150 amino acid residues between residues 1- 202 of SEQ ID NO: 2. In still more preferred embodiments the nucleic acid sequence includes at least about 1 to 100, preferably about 2 to 250, more preferably about 3 to 20, still more preferably about 5 to 10 nucleotides from the region of SEQ ID NO: 1 which encodes amino acid residues 1-202 of SEQ ID NO: 2. The encoded peptide includes at least about 1 to 30, preferably about 2 to 20, more preferably about 5 to 10 amino acid residues from amino acid residues 1-202 of SEQ ID NO: 2. The CR2 gene comprises 1283 nucleotides, which is a fragment of SEQ. ID No: 3 encoding a small, intracellular protein of 60 amino acids (about 6.6 kD), of SEQ. ID No: 4. The CR2 gene is the only CR gene for which there are no homologies to other known gene products. Elevated cAMP suppresses, but does not abolish CR2 gene expression. The obtained DNA sequence and the deduced protein sequence for CR2 are provided below in Table III. Table III: Full DNA Sequence and Deduced Protein Sequence for CR2
ATTTAGAGCA ACTCAGGAAA TAGGTGCACA CAAGCAAACC ATGTGGTTAA AGCCTTTGGA 60
ACTGGTTTGA GCAAAGCTGT AGGTGATTTG ACAAAATCAT CTGCAAAACC AGATTTCTAA 120 CACCTCCCTG CTGTGTATCT CATTTCTGCT GATGTGTGGT GCTTCATAAG ATG GGG 176
Met Gly 1
ACG TTA AGC ATG CAG CAA CTA CAG TCA TTT GTT CTC AGA GGT CTG GAC 224 Thr Leu Ser Met Gin Gin Leu Gin Ser Phe Val Leu Arg Gly Leu Asp 5 10 15
CAA AGA GAA ACA AGA AAA GCT GGA GTC ACA CTA CCA AAG GCC GAA GCT 272 Gin Arg Glu Thr Arg Lys Ala Gly Val Thr Leu Pro Lys Ala Glu Ala
20 25 . 30
GAG CAA CAG AGC TCT GGA GTC AGC TGC CTG GGT TCA GCA TGC AGC GCT 320 Glu Gin Gin Ser Ser Gly Val Ser Cys Leu Gly Ser Ala Cys Ser Ala 35 40 45 50
GCC GTG GAC GAT CTG- TCT CTC TTG CAT ATA T GACTTACCAG TTTTACTTTC 371 Ala Val Asp Asp Leu Ser Leu Leu His lie (SEQ. ID No: 4) 55 60 AGTCTCTCCA TTTCTAATTA AATGAGATGC AGAAATGCTG GTGCCTTGCT ATGATGTTTG 431
CAGTTATTAT TTCTAGGAAA AAAAATATTA TTGTTACTCA GTATCTGGTC TAGCTACTTG 491
GACAACTGGA CTATCCCCCT CCTTTCAAGG GAGGGCAAAG CATTTCAGAA AAGAACTAAG 551
TGCTATTTCT CTGCTTCAGG AATGTCTCCC GTATGTAAAA GAATGTGGCT TCAGGGAGTA 611
GCATGTGTTG TAAAGGTGGA TGGGTCTAAC TTCATGGACA GCTCTGACAT CCACTAGCTA 671 TGCCACCTGA TGCAAACCAC TTGGGCTGTC TGCAGTTTCG TTTATCTTTC TGGAATTGGT 731
AATAACAACC ACCTGGCAAG ATCACTGTTA TGAATACGGA GGATCAAAGT TGTGAAGTTA 791
TTTTGTAAAG TGAAATGTTC TGAAAAATGG ATTTTAACAG TGTCAGCGAA AAGTAGATTT 851
TTGACATTTA TCAAGAGTTC AGCTAATGAA AACAAGTATG GATAATAGTT ACATAGAACT 911
GTCTACTTTA CTCAGTACTT TAGCATATGC TATTATATTT AATCTTCTTA AAAAGTAGGA 971 AATTATACAA GCCATGTATT GATATTATTG TGGTGGTTGT CGTTCTCAAT TACACACTGA 1031
ATATTAAGAC CTCTCAGGTA GCAGCTGGAA GGACATTGTA TCCAGTTTCC TGATTGTTTT 1091
CAATGGAATA ATCATGTATA CATGCACTAC TAATGAGACA ATGGTGATTC TAAAAGCTTA 1151
ATCAGGGGGA CTTTTGTGTA TTCCAAATCT ACTAAAAATA AAGAAACACA GAAATGAGAA 1211
AAAAAAAAAA AA (SEQ. ID No: 3) 1223
The present invention also pertains to a CR2 polypeptide, preferably a substantially pure preparation of a CR2 polypeptide, or a recombinant CR2 polypeptide. In preferred embodiments, the CR2 polypeptide comprises an amino acid sequence at least about 60% to 95%, preferably about 80% to 90% homologous to the amino acid sequence in SEQ ID NO:4, essentially the same as the SEQ ID NO:4, or fragments at least about 5 to 250, preferably about 10 to 100, and more preferably about 20 to 50 amino acids in length, at least about 5, preferably at least about 10, and more preferably at least about 20, 50, 100, or 150 contiguous amino acids from SEQ ID NO: 4. In further preferred embodiments, a protein homologous to SEQ ID NO: 4 has a molecular weight in the range of about 5-15 kD, and more preferably about 6 kD. In a preferred embodiment, a polypeptide having at least one biological activity of the CR2 polypeptide may differ in amino acid sequence from the sequence in SEQ ID NO: 4, but such differences result in a modified polypeptide which funαions in the same or similar manner as native CR2 protein or which has the same or similar characteristics of the native CR2 protein. Such a peptide can include at least about 1 to 30, preferably about 2 to 20, and more preferably about 5 to 10 amino acid residues from residues 1-60 of SEQ ID NO: 4. In yet other preferred embodiments, the CR2 polypeptide is a recombinant fusion protein which comprises a second polypeptide portion, e.g., a second polypeptide comprising an amino acid sequence unrelated to a protein represented by SEQ ID NO: 4, e.g., glutathione-S-transferase, a DNA binding domain, a polymerase aαivating domain, so that the fusion protein is functional in a two-hybrid assay. Yet another aspeα of the present invention concerns an immunogen comprising a CR2 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the CR2 polypeptide; e.g. a humoral, antibody, or cellular response. In preferred embodiments, the immunogen comprises an antigenic determinant, e.g. a unique determinant, from the SEQ ID NO: 4. A further aspect of the present invention features a monoclonal or polyclonal antibody preparation specifically reactive with an epitope of the CR2 immunogen.
Another aspect of the present invention provides a substantially pure nucleic acid comprising a nucleotide sequence which encodes a CR2 polypeptide, analogues and/or fragments thereof. Furthermore, in certain preferred embodiments, the subjeα CR2 nucleic acid includes a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the CR2 gene sequence, e.g., to render the CR2 gene sequence suitable for use as an expression veαor. In yet a further preferred embodiment, the nucleic acid which encodes a CR2 polypeptide hybridizes under stringent conditions to a nucleic acid probe corresponding to at least about 12 to 60, more preferably to at least about 20 to 50, still more preferably to at least about 40 to 45 consecutive nucleotides of SEQ ID NO: 3. In yet a further preferred embodiment, the CR2 encoding nucleic acid hybridizes to a nucleic acid probe corresponding to a subsequence encoding at least about 4 to 30, more preferably at least about 10 to 25 consecutive amino acid residues, and even more preferably at least about 20 amino acid residues between residues 1-60 of SEQ ID NO: 4. In preferred embodiments the nucleic acid sequence includes at least about 1 to 100, preferably about 2 to 50, more preferably about 3 to 30, and still more preferably about 5 to 20 nucleotides from die region of SEQ ID NO: 3 which encodes amino acid residues 1-60 of SEQ ID NO: 4. The encoded peptide includes at least about 1 to 30, preferably about 2 to 20, and more preferably about 3 to 10 amino acid residues 1-60 of SEQ ID NO: 4.
Gene and polypeptide analogues and fragments of the CR2 gene and encoded polypeptide and anologues are also pan of this invention and take the form described above in relation to CRI. The CR3 gene includes 2451 nucleotides, shown as the fragment of SEQ. ID NO: 5 encoding a protein of 378 amino acids (about 41.5 kD) of SEQ. ID NO: 6. The corresponding DNA and protein sequences corresponding to CR3 are shown in Table IV below.
Table IV: Full DNA and Protein Sequences for CR3
CGCGGGAGCC TCGAGCGCCG CTCGGATGCA GAAGCCGAGC CGCCACTCGG CGCGCGGTGG 60
GAGACCCAGG GCAAGCCGCC GTCGGCGCGC TGGGTGCGGG AAGGGGGCTC TGGATTTCGG 120 TCCCTCCCCT TTTTCCTCTG AGTCTCGGAA CGCTCCAGAT CTCAGACCCT CTTCCTCCCA 180
GGTAAAGGCC GGGAGAGGAG GGCGCATCTC TTTTCCAGGC ACCCCACC ATG GGA AAT 237
Met Gly Asn 1 GCC TCC AAT GAC- TCC CAG TCT GAG GAC TGC GAG ACG CGA CAG TGG TTT 285 Ala Ser Asn Asp Ser Gin Ser Glu Asp Cys Glu Thr Arg Gin Trp Phe 5 10 15
CCC CCA GGC GAA AGC CCA GCC ATC AGT TCC GTC ATG TTC TCG GCC GGG 333 Pro Pro Gly Glu Ser Pro Ala lie Ser Ser Val Met Phe Ser Ala Gly 20 25 30 35 GTG CTG GGG AAC CTC ATA GAA CTG GCG CTG CTG GCG CGC CGC TGG CAG 381 Val Leu Gly Asn Leu lie Glu Leu Ala Leu Leu Ala Arg Arg Trp Gin
40 45 50
GGG GAC GTG GGG TGC AGC GCC GGC CGT AGG AGC TCC CTC TCC TTG TTC 429 Gly Asp Val Gly Cys Ser Ala Gly Arg Arg Ser Ser Leu Ser Leu Phe 55 60 65
CAC GTG CTG GTG ACC GAG CTG GTG TTC ACC GAC CTG CTC GGG ACC TGC 477 His Val Leu Val Thr Glu Leu Val Phe Thr Asp Leu Leu Gly Thr Cys
70 75 80
CTC ATC AGC CCA GTG GTA CTG GCT TCG TAC GCG CGG AAC CAG ACC CTG 525 Leu lie Ser Pro Val Val Leu Ala Ser Tyr Ala Arg Asn Gin Thr Leu 85 90 95
GTG GCA CTG GCG CCC GAG AGC CGC GCG TCC ACC TAC TTC GCT TTC GCC 573 Val Ala Leu Ala Pro Glu Ser Arg Ala Ser Thr Tyr Phe Ala Phe Ala 100 105 110 115 ATG ACC TTC TTC AGC CTG GCC ACG ATG CTC ATG CTC TTC ACC ATG GCC 621 Met Thr Phe Phe Ser Leu Ala Thr Met Leu Met Leu Phe Thr Met Ala
120 125 130
CTG GAG CGC TAC CTC TCG ATC GGG CAC CCC TAC TTC TAC CAG CGC CGC 669 Leu Glu Arg Tyr Leu Ser lie Gly His Pro Tyr Phe Tyr Gin Arg Arg 135 140 145
GTC TCG CGC TCC GGG GGC CTG GCC GTG CTG CCT GTC ATC TAT GCA GTC 717 Val Ser Arg Ser Gly Gly Leu Ala Val Leu Pro Val lie Tyr Ala Val
150 155 160
TCC CTG CTC TTC TGC TCA CTG CCG CTG CTG GAC TAT GGG CAG TAC GTC 765 S Seerr L Leeuu L Leeuu Phe Cys Ser Leu Pro Leu Leu Asp Tyr Gly Gin Tyr Val 165 170 175
CAG TAC TGC CCC GGG ACC TGG TGC TTC ATC C CGGGG C CAACC G GGGGG C CGGGG A ACCCC G GCCTT 813
Gin Tyr Cys Pro Gly Thr Trp Cys Phe lie Arg His Gly Arg Thr Ala
180 185 190 195 T TAACC C CTTGG C CAAGG CTG TAC GCC ACC CTG CTG CTG C CTTTT C CTTCC A ATTTT G GTTCC T TCCGG G GTTGG 861 Tyr Leu Gin Leu Tyr Ala Thr Leu Leu Leu Leu Leu He Val Ser Val 200 205 210
CTC GCC TGC AAC TTC AGT GTC ATT CTC AAC C CTTCC A ATTCC C CGGCC A ATTGG C CAACC C CGGCC 909 Leu Ala Cys Asn Phe Ser Val lie Leu Asn Leu He Arg Met His Arg 215 220 225 TableJV: Full DNA and Protein Sequences for CR3 (Cont'd)
CGA AGC CGG AGA AGC CGC TGC GGA CCT TCC CTG GGC AGT GGC CGG GGC 957 Arg Ser Arg Arg Ser Arg Cys Gly Pro Ser Leu Gly Ser Gly Arg Gly 230 235 240
GGC CCC GGG GCC CGC AGG AGA GGG GAA AGG GTG TCC ATG GCG GAG GAG 1005 Gly Pro Gly Ala Arg Arg Arg Gly Glu Arg Val Ser Met Ala Glu Glu
245 250 255
ACG GAC CAC CTC ATT CTC CTG GCT ATC ATG ACC ATC ACC TTC GCC GTC 1053 Thr Asp His Leu He Leu Leu Ala He Met Thr He Thr Phe Ala Val 260 265 270 275
TGC TCC TTG CCT TTC ACG ATT TTT GCA TAT ATG AAT GAA ACC TCT TCC 1101 Cys Ser Leu Pro Phe Thr He Phe Ala Tyr Met Asn Glu Thr Ser Ser 280 285 290 CGA AAG GAA AAA TGG GAC CTC CAA GCT CTT AGG TTT TTA TCA ATT AAT 1149 Arg Lys Glu Lys Trp Asp Leu Gin Ala Leu Arg Phe Leu Ser He Asn
295 300 305
TCA ATA ATT GAC CCT TGG GTC TTT GCC ATC CTT AGG CCT CCT GTT CTG 1197 Ser He He Asp Pro Trp Val Phe Ala He Leu Arg Pro Pro Val Leu 310 315 320
AGA CTA ATG CGT TCA GTC CTC TGT TGT CGG ATT TCA TTA AGA ACA CAA 1245 Arg Leu Met Arg Ser Val Leu Cys Cys Arg He Ser Leu Arg Thr Gin
325 330 335
GAT GCA ACA CAA ACT TCC TGT TCT ACA CAG TCA GAT GCC AGT AAA CAG 1293 Asp Ala Thr Gin Thr Ser Cys Ser Thr Gin Ser Asp Ala Ser Lys Gin 340 345 350 355
GCT GAC CTT T GAGGTCAGTA GTTTAAAAGT TCTTAGTTAT ATAGCATCTG 1343
Ala Asp Leu (SEQ. ID No: 6)
357 GAAGATCATT TTGAAATTGT TCCTTGGAGA AATGAAAACA GTGTGTAAAC AAAATGAAGC 1403 TGCCCTAATA AAAAGGAGTA TACAAACATT TAAGCTGTGG TCAAGGCTAC AGATGTGCTG 1463 ACAAGGCACT TCATGTAAAG TGTCAGAAGG AGCTACAAAA CCTACCCTCA GTGAGCATGG 1523 TACTTGGCCT TTGGAGGAAC AATCGGCTGC ATTGAAGATC CAGCTGCCTA TTGATTTAAG 1583 CTTTCCTGTT GAATGACAAA GTATGTGGTT TTGTAATTTG TTTGAAACCC CAAACAGTGA 1643 CTGTACTTTC TATTTTAATC TTGCTACTAC CGTTATACAC ATATAGTGTA CAGCCAGACC 1703 AGATTAAACT TCATATGTAA TCTCTAGGAA GTCAATATGT GGAAGCAACC AAGCCTGCTG 1763 TCTTGTGATC ACTTAGCGAA CCCTTTATTT GAACAATGAA GTTGAAAATC ATAGGCACCT 1823 TTTACTGTGA TGTTTGTGTA TGTGGGAGTA CTCTCATCAC TACAGTATTA CTCTTACAAG 1883 AGTGGACTCA GTGGGTTAAC ATCAGTTTTG TTTACTCATC CTCCAGGAAC TGCAGGTCAA 1943 GTTGTCAGGT TATTTATTTT ATAATGTCCA TATGCTAATA GTGATCAAGA AGACTTTAGG 2003 AATGGTTCTC TCAACAAGAA ATAATAGAAA TGTCTCAAGG CAGTTAATTC TCATTAATAC 2063 TCTTTATCCT ATTTCTGGGG GAGGATGTAC GTGGCCATGT ATGAAGCCAA ATATTAGGCT 2123 TAAAAACTGA AAAATCTGGT TCATTCTTCA GATATACTGG AACCCTTTTA AAGTTGATAT 2183 TGGGGCCATG AGTAAAATAG ATTTTATAAG ATGACTGTGT TGTACTAAAA TTCATCTGTC 2243 TATATTTTAT TTAGGGGACA TGGTTTGACT CATCTTATAT GGGAAACCAT GTAGCAGTGA 2303 GTCATATCTT AATATATTTC TAAATGTTTG GCATGTAAAC GTAAACTCAG CATCACAATA 2363 TTTCAGTGAA TTTGCACTGT TTAATCATAG TTACTGTGTA AACTCATCTG AAATGTTACC 2423 AAAAATAAAC TATAAAACAA AATTTGA (SEQ ID No: 5) 2450
The present invention further pertains to a CR3 polypeptide, preferably a substantially pure preparation of a CR3 polypeptide, or a recombinant CR3 polypeptide. In preferred embodiments, the CR3 polypeptide comprises an amino acid sequence at least about 60% to 95%. preferably about 80% to 90% homologous to the SEQ ID NO: 6. The polypeptide comprises an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NO: 6, and comprises at least about 5 to 150, preferably about 10 to 100, and more preferably about 20 to 50 amino acids in length. The polypeptide comprises at least about 5, preferably at least about 10, more preferably at least about 20, 50, still more preferably at least about 100, or 150 contiguous amino acids from SEQ ID NO: 6. In further preferred embodiments, a protein homologous to SEQ ID NO: 6 has a molecular weight in the range of about 80-95 kD, preferably about 88 . In a preferred embodiment, a peptide having at least one biological aαivity of the CR3 polypeptide may differ in amino acid sequence from the sequence in SEQ ID NO: 6, but such differences result in a modified protein which functions in me same or similar manner as or which has the same or similar characteristics of native CR3 protein. Such a peptide can include at least about 1, 2, 3, or 5, and preferably about 10, 20, and 30, amino acid residues from residues 1-358 of SEQ ID NO: 6.
In yet other preferred embodiments, the CR3 polypeptide is a recombinant fusion protein which comprises a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated to the SEQ ID NO: 6, e.g., the second polypeptide portion is glutathione-S- transferase, a DNA binding domain, a polymerase aαivating domain, e.g., me fusion protein is funαional in a two-hybrid assay. Yet anod er aspeα of the present invention concerns an immunogen comprising a CR3 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for said CR3 polypeptide, e.g. a humoral response, an antibody response, or a cellular response. In preferred embodiments, the immunogen comprises an antigenic determinant, e.g. a unique determinant from the SEQ ID NO: 6. A further aspect of the present invention features a monoclonal or polyclonal antibody preparation specifically reactive with an epitope of the CR3 immunogen.
Another aspect of the present invention provides a substantially pure nucleic acid comprising a nucleotide sequence which encodes a CR3 polypeptide. In certain preferred embodiments, me subject CR3 nucleic acid includes a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the CR3 gene sequence, e.g., to render the CR3 gene sequence suitable for use as an expression vector. In yet a further preferred embodiment, the nucleic acid which encodes a CR3 polypeptide hybridizes under stringent conditions to a nucleic acid probe corresponding to at least about 12 to 300, more preferably to at least about 20 to 200, and still more preferably to at least about 40 to 100 consecutive nucleotides of SEQ ID NO: 5. In yet a further preferred embodiment, the CR3 encoding nucleic acid hybridizes to a nucleic acid probe corresponding to a subsequence encoding at least about 4, more preferably at least about 10, and even more preferably at least about 20 amino acid residues between residues 1-358 of SEQ ID NO: 6. In preferred embodiments the nucleic acid sequence includes at least about 1, 2, 3 or 5, and preferably at least about 10, 20, 50, or 100 nucleotides from the region of SEQ ID NO: 5, which encodes amino acid residues 1-358 of SEQ ID NO: 6. The encoded peptide includes at least about 1, 2, 3, 5, 10, 20, or 30 amino acid residues from amino acid residues 1-358 of SEQ ID NO: 6. This protein has some homology to the G-coupled, 7 transmembrane-spanning receptors of the prostaglandin family. The receptor for prostacyclin (PGI2) is most homologous (about 70%) (See, Boie, Y. et al., J. Biol. Chem. 269: 12173-12178 (1994)) to the CR3 protein. PGI2 is a labile metabolite of arachidonic acid produced via the cyclooxygenase pathway, and plays a major physiological role as a potent mediator of vasodilation and inhibitor of platelet activation. It is primarily expressed in the kidney with lower levels of mRNA also observed in the lung and the liver. In the kidney,, the PGI2 receptor is thought to play an important role in renal blood flow, renin release, and glomerular filtration rate. By comparison, CR3 is maximally expressed by leukocytes, placenta, testes, ovary and small intestine, and at lower levels by spleen, thymus and prostate, but not by kidney or liver. CR3, most likely thus, plays a regulatory role in cellular proliferation and or inflammation. Elevated cAMP suppresses CR3 expression early on after IL-2 stimulation, but not at a later time. Because the protein encoded by CR3 is a member of a family of 7 transmembrane spanning receptors, this receptor is likely coupled to cytoplasmic GTP- binding proteins (G-proteins), that are known to activate or suppress the generation of cAMP. The CR3 gene product, therefore, provides a new receptor that allows the manipulation of cellular functions controlled by biochemical pathways signaled by the receptor. The CR3 gene and gene product may be used in assays for identifying other ligands that trigger the receptor. These ligands can be used to modulate cellular proliferation and inflammation.
DNA and polypeptide analogues and fragment analogues and original DNA and polypeptide CR3 sequences are also pan of the invention, in the form described above for CRI. The CR4 gene comprises a 2946 nucleotide fragment of SEQ. ID NO: 7, which encodes a protein of 763 amino acids (about 85.9 kD), of SEQ. ID NO: 8. These sequences are shown in Table V below.
Table V: Full DNA and Deduced Protein Sequence for CR4
GGGGGGAAAG GAAAATAATA CAATTTCAGG GGAAGTCGCC TTCAGGTCTG CTGCTTTTTT 60
ATTTTTTTTT TTTTAATTAA AAAAAAAAAG GACATAGAAA ACATCAGTCT TGAACTTCTC 120
TTCAAGAACC CGGGCTGCAA AGGAAATCTC CTTTGTTTTT GTTATTTATG TGCTGTCAAG 180
TTTTGAAGTG GTGATCTTTA GACAGTGACT GAGT ATG GAT CAT TTG AAC GAG 232
Met ASD His Leu Asn Glu 1 " 5 Table V: Full DNA and Deduced Protein Sequence for CR4 (Cont'd)
GCA ACT CAG GGG AAA GAA CAT TCA GAA ATG TCT AAC AAT GTG AGT GAT 280
Ala Thr Gin Gly Lys Glu His Ser Glu Met Ser Asn Asn Val Ser Asp 10 15 20
CCG AAG GGT CCA CCA GCC AAG ATT GCC CGC CTG GAG CAG AAC GGG AGC 328
Pro Lys Gly Pro Pro Ala Lys He Ala Arg Leu Glu Gin Asn Gly Ser 25 30 35
CCG CTA GGA AGA GGA AGG CTT GGG AGT ACA GGT GCA AAA ATG CAG GGA 376
Pro Leu Gly Arg Gly Arg Leu Gly Ser Thr Gly Ala Lys Met Gin Gly
40 45 50
GTG CCT TTA AAA CAC TCG GGC CAT CTG ATG AAA ACC AAC CTT AGG AAA 424
Val Pro Leu Lys His Ser Gly His Leu Met Lys Thr Asn Leu Arg Lys
55 60 65 70
GGA ACC ATG CTG CCA GTT TTC TGT GTG GTG GAA CAT TAT GAA AAC GCC 472
Gly Thr Met Leu Pro Val Phe Cys Val Val Glu His Tyr Glu Asn Ala . 75 80 85
ATT GAA TAT GAT TGC' AAG GAG GAG CAT GCA GAA TTT GTG CTG GTG AGA 520
He Glu Tyr Asp Cys Lys Glu Glu His Ala Glu Phe Val Leu Val Arg 90 95 100
AAG GAT ATG CTT TTC AAC CAG CTG ATC GAA ATG GCA TTG CTG TCT CTA 568
Lys Asp Met Leu Phe Asn Gin Leu He Glu ■Met Ala Leu Leu Ser Leu 105 110 115
GGT TAT TCA CAT AGC TCT GCT GCC CAG GCC AAA GGG CTA ATC CAG GTT 616
Gly Tyr Ser His Ser Ser Ala Ala Gin Ala Lys Gly Leu He Gin Val 120 125 130
GGA AAG TGG AAT CCA GTT CCA CTG TCT TAC GTG ACA GAT GCC CCT GAT 664
Gly Lys Trp Asn Pro Val Pro Leu Ser Tyr Val Thr Asp Ala Pro Asp
135 140 145 150
GCT ACA GTA GCA GAT ATG CTT CAA GAT GTG TAT CAT GTG GTC ACA TTG 712
Ala Thr Val Ala Asp Met Leu Gin Asp Val Tyr His Val Val Thr Leu 155 160 165
AAA ATT CAG TTA CAC AGT TGC CCC AAA CTA GAA GAC TTG CCT CCC GAA 760
Lys He Gin Leu His Ser Cys Pro Lys Leu Glu Asp Leu Pro Pro Glu 170 175 180
CAA TGG TCG CAC ACC ACA GTG AGG AAT GCT CTG AAG GAC TTA CTG AAA 808
Gin Trp Ser His Thr Thr Val Arg Asn Ala Leu Lys Asp Leu Leu Lys 185 190 195
GAT ATG AAT CAG AGT TCA TTG GCC AAG GAG TGC CCC CTT TCA CAG AGT 856
Asp Met Asn Gin Ser Ser Leu Ala Lys Glu Cys Pro Leu Ser Gin Ser 200 205 210
ATG ATT TCT TCC ATT GTG AAC AGT ACT TAC TAT GCA AAT GTC TCA GCA 904
Met He Ser Ser He Val Asn Ser Thr Tyr Tyr Ala Asn Val Ser Ala
215 220 225 230
GCA AAA TGT CAA GAA TTT GGA AGG TGG TAC AAA CAT TTC AAG AAG ACA 952
Ala Lys Cys Gin Glu Phe Gly Arg Trp Tyr Lys His Phe Lys Lys Thr
235 240 245
AAA GAT ATG ATG GTT GAA ATG GAT AGT CTT TCT GAG CTA TCC CAG CAA 1000
Lys Asp Met Met Val Glu Met Asp Ser Leu Ser Glu Leu Ser Gin Gin 250 255 260
GGC GCC AAT CAT GTC AAT TTT GGC CAG CAA CCA GTT CCA GGG AAC ACA 1048
Gly Ala Asn His Val Asn Phe Gly Gin Gin Pro Val Pro Gly Asn Thr 265 270 275
GCC GAG CAG CCT CCA TCC CCT GCG CAG CTC TCC CAT GGC AGC CAG CCC 1096
Ala Glu Gin Pro Pro Ser Pro Ala Gin Leu Ser His Gly Ser Gin Pro 280 285 290 Table V: Full DNA and Deduced Protein Sequence for CR4 (Cont'd)
TCT GTC CGG ACA CCT CTT CCA AAC CTG CAC CCT GGG CTC GTA TCA ACA 1144 Ser Val Arg Thr Pro Leu Pro Asn Leu His Pro Gly Leu Val Ser Thr 295 300 305 310
CCT ATC AGT CCT CAA TTG GTC AAC CAG CAG CTG GTG ATG GCT CAG CTG 1192 Pro He Ser Pro Gin Leu Val Asn Gin Gin Leu Val Met Ala Gin Leu
315 320 325
CTG AAC CAG CAG TAT GCA GTG AAT AGA CTT TTA GCC CAG CAG TCC TTA 1240 Leu Asn Gin Gin Tyr Ala Val Asn Arg Leu Leu Ala Gin Gin Ser Leu
330 335 340
AAC CAA CAA TAC TTG AAC CAC CCT CCC CCT GTC AGT AGA TCT ATG AAT 1288
Asn Gin Gin Tyr Leu Asn His Pro Pro Pro Val Ser Arg Ser Met Asn 345 350 355
AAAAGG CCCCTT TTTTGG GGAAGG-- CCAAAA CCAAGG GGTTTT TTCCGG AACCCC AAAACC AACCAA GGAAGG GGTTGG TTCCTT TTCCCC GGAAAA 1336
Lys Pre Leu Glu Gin Gin Val Ser Thr Asn Thr Glu Val Ser Ser Glu 360 365 370
ATC TAC CAG TGG GTA CGC GAT GAA CTG AAA CGA GCA GGA ATC TCC CAG 1384
He Tyr Gin Trp Val Arg Asp Glu Leu Lys Arg Ala Gly He Ser Gin 375 380 385 390
GCG GTA TTT GCA CGT GTG GCT TTT AAC AGA ACT CAG GGC TTG CTT TCA 1432 Ala Val Phe Ala Arg Val Ala Phe Asn Arg Thr Gin Gly Leu Leu Ser
395 400 405
GAA ATC CTC CGA AAG GAA GAG GAC CCC AAG ACT GCA TCC CAG TCT TTG 1480 Glu He Leu Arg Lys Glu Glu Asp Pro Lys Thr Ala Ser Gin Ser Leu 410 415 420
CTG GTA AAC CTT CGG GCT ATG CAG AAT TTC TTG CAG TTA CCG GAA GCT 1528 Leu Val Asn Leu Arg Ala Met Gin Asn Phe Leu Gin Leu Pro Glu Ala 425 430 435 GAA AGA GAC CGA ATA TAC CAG GAC GAA AGG GAA AGG AGC TTG AAT GCT 1576 Glu Arg Asp Arg He Tyr Gin Asp Glu Arg Glu Arg Ser Leu Asn Ala
440 445 450
GCC TCG GCC ATG GGT CCT GCC CCC CTC ATC AGC ACA CCA CCC AGC CGT 1624 Ala Ser Ala Met Gly Pro Ala Pro Leu He Ser Thr Pro Pro Ser Arg 455 460 465 470
CCT CCC CAG GTG AAA ACA GCT ACT ATT GCC ACT GAA AGG AAT GGG AAA 1672 Pro Pro Gin Val Lys Thr Ala Thr He Ala Thr Glu Arg Asn Gly Lys
475 480 485
CCA GAG AAC AAT ACC ATG AAC ATT AAT GCT TCC ATT TAT GAT GAG ATT 1720 Pro Glu Asn Asn Thr Met Asn He Asn Ala Ser He Tyr Asp Glu He 490 495 500
CAG CAG GAA ATG AAG CGT GCT AAA GTG TCT CAA GCA CTG TTT GCA AAG 1768 Gin Gin Glu Met Lys Arg Ala Lys Val Ser Gin Ala Leu Phe Ala Lys 505 510 515 GTT GCA GCA ACC AAA AGC CAG GGA TGG TTG TGC GAG CTG TTA CGC TGG 1816 Val Ala Ala Thr Lys Ser Gin Gly Trp Leu Cys Glu Leu Leu Arg Trp
520 525 530
AAA GAA GAT CCT TCT CCA GAA AAC AGA ACC CTG TGG GAG AAC CTC TCC 1864 Lys Glu Asp Pro Ser Pro Glu Asn Arg Thr Leu Trp Glu Asn Leu Ser 535 540 545 550
ATG ATC CGA AGG TTC CTC AGT CTT CCT CAG CCA GAA CGT GAT GCC ATT 1912 Met He Arg Arg Phe Leu Ser Leu Pro Gin Pro Glu Arg Asp Ala He
555 560 565
TAT GAA CAG GAG AGC AAC GCG GTG CAT CAC CAT GGC GAC AGG CCG CCC 1960 Tyr Glu Gin Glu Ser Asn Ala Val His His His Gly Asp Arg Pro Pro 570 575 580 Table V: Full DNA and Deduced Protein Sequence for CR4 (Cont'd)
CAC ATT ATC CAT GTT CCA GCA GAG CAG ATT CAG CAA CAG CAG CAG CAA 2008 His He He His Val Pro Ala Glu Gin He Gin Gin Gin Gin Gin Gin 585 590 595
CAG CAA CAG CAG CAG CAG CAG CAG CAG GCA CCG CCG CCT CCA CAG CCA 2056 Gin Gin Gin Gin Gin Gin Gin Gin Gin Ala Pro Pro Pro Pro Gin Pro
600 605 610
CAG CAG CAG CCA CAG ACA GGC CCT CGG CTC CCC CCA CGG CAA CCC ACG 2104 Gin Gin Gin Pro Gin Thr Gly Pro Arg Leu Pro Pro Arg Gin Pro Thr 615 620 625 630
GTG GCC TCT CCA GCA GAG TCA GAT GAG GAA AAC CGA CAG AAG ACC CGG 2152 Val Ala Ser Pro Ala Glu Ser Asp Glu Glu Asn Arg Gin Lys Thr Arg 635 640 645 CCA CGA ACA AAA ATT TCA GTG GAA GCC TTG GGA ATC CTC CAG AGT TTC 2200 Pro Arg Thr Lys He Ser Val Glu Ala Leu Gly He Leu Gin Ser Phe
650 655 660
ATA CAA GAC GTG GGC CTG TAC CCT GAC GAA GAG GCC ATC CAG ACT CTG 2248 He Gin Asp Val Gly Leu Tyr Pro Asp Glu Glu Ala He Gin Thr Leu 665 670 675
TCT GCC CAG CTC GAC CTT CCC AAG TAC ACC ATC ATC AAG TTC TTT CAG 2296 Ser Ala Gin Leu Asp Leu Pro Lys Tyr Thr He He Lys Phe Phe Gin
680 685 690
AAC CAG CGG TAC TAT CTC AAG CAC CAC GGC AAA CTG AAG GAC AAT TCC 2344 Asn Gin Arg Tyr Tyr Leu Lys His His Gly Lys Leu Lys Asp Asn Ser 695 700 705 710
GGT TTA GAG GTC GAT GTG GCA GAA TAT AAA GAA GAG GAG CTG CTG AAG 2392 Gly Leu Glu Val Asp Val Ala Glu Tyr Lys Glu Glu Glu Leu Leu Lys 715 720 725 GAT TTG GAA GAG AGT GTC CAA GAT AAA AAT ACT AAC ACC CTT TTT TCA 2440 Asp Leu Glu Glu Ser Val Gin Asp Lys Asn Thr Asn Thr Leu Phe Ser
730 735 740
GTG AAA CTA GAA GAA GAG CTG TCA GTG GAA GGA AAC ACA GAC ATT AAT 2488 Val Lys Leu Glu Glu Glu Leu Ser Val Glu Gly Asn Thr Asp He Asn 745 750 755
ACT GAT TTG AAA GAC TGAGATAAAA GTATTTGTTT CGTTCAACAG TGCCACTGGT 2543 Thr Asp Leu Lys Asp (SEQ. ID No: 8)
760 ATTTACTAAC AAAATGAAAA GTCCACCTTG TCTTCTCTCA GAAAACCTTT GTTGTTCATT 2603 GTTTGGCCAA TGAACTTTCA AAAACTTGCA CAAACAGAAA AGTTGGAAAA GGATAATACA 2663 GACTGCACTA AATGTTTTCC TCTGTTTTAC AAACTGCTTG GCAGCCCCAG GTGAAGCATC 2723 AAGGATTGTT TGGTATTAAA ATTTGTGTTC ACGGGATGCA CCAAAGTGTG TACCCCGTAA 2783 GCATGAAACC AGTGTTTTTT GTTTTTTTTT TAGTTCTTAT TCCGGAGCCT CAAACAAGCA 2843 TTATACCTTC TGTGATTATG ATTTCCTCTC CTATAATTAT TTCTGTAGCA CTCCACACTG 2903 ATCTTTGGAA ACTTGCCCCT TATTTAAAAA AAAAAAAAAA AAA (SEQ. ID No: 7) 2946
The present invention still further pertains to a CR4 polypeptide, preferably a substantially pure preparation of a CR4 polypeptide, or a recombinant CR4 polypeptide. In preferred embodiments, the CR4 polypeptide comprises an amino acid sequence at least about 60% to 95%, preferably about 80% to 90%, homologous to the amino acid sequence in SEQ ID NO: 8, essentially the same as the amino acid SEQ ID NO: 8, comprising at least about 5 to 150, preferably about 10 to 100. more preferably about 20 to 50 amino acids in length, comprises at least about 5 to 150, preferably at least about 10 to 100, and more preferably at least about 20 to 50 contiguous amino acids from SEQ ID NO: 8. In further preferred embodiments, a protein homologous to SEQ ID NO: 8 has a molecular weight in the range of about 75-90 kD, preferably about 83 . In a preferred embodiment, a polypeptide having at least one biological aαivity of the CR4 polypeptide may differ in amino acid sequence from me sequence in SEQ ID NO:8, but such differences result in a modified polypeptide which functions in the same or similar manner as native CR4 protein or which has the same or similar characteristics of the native CR4 protein. Such a peptide can include at least about 1 to 30, preferably about 2 to 20, and more preferably about 3 to 10, or about 5 amino acid residues from residues 1-763 of SEQ ID NO: 8. In yet other preferred embodiments, the CR4 polypeptide is a recombinant fusion protein which comprises a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated to a protein represented by SEQ ID NO: 8, e.g., the second polypeptide portion is glutathione-S-transferase, a DNA binding domain, a polymerase aαivating domain, e.g., the fusion protein is funαional in a two-hybrid assay. Yet another aspect of the present invention pertains to an immunogen comprising a CR4 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the CR4 polypeptide; e.g. a humoral response, an antibody response; or a cellular response. In preferred embodiments, the immunogen comprises an antigenic determinant, e.g. a unique determinant, comprising SEQ ID NO: 8. A further aspect of the present invention features a monoclonal or polyclonal antibody preparation specifically reactive with an epitope of the CR4 immunogen.
Another aspeα of the present invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes a CR4 polypeptide, analogues and/or fragments thereof. In certain preferred embodiments, the CR4 nucleic acid comprises a transcriptional regulatory sequence, e.g., at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the CR4 gene sequence, e.g., to render the CR4 gene sequence suitable for use as an expression veαor. In yet a further preferred embodiment, the nucleic acid which encodes a CR4 polypeptide of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least about 12 to 300, more preferably to at least about 20 to 200, more preferably to at least about 40 to 100 consecutive nucleotides of SEQ ID NO: 7. In yet a further preferred embodiment, the CR4 nucleic acid hybridizes to a nucleic acid probe corresponding to a subsequence encoding at least about 4 to 60, more preferably at least about 10 to 40, and even more preferably at least about 20 to 30 consecutive amino acid residues between residues 1-763 of SEQ ID NO: 8. In preferred embodiments, the nucleic acid sequence comprises at least about 1, 2, 3 or 5, and preferably at least about 10. 20, 50, or 100 nucleotides from the region of SEQ ID NO: 7 which encodes amino acid residues 1-763 of SEQ ID NO: 8. The encoded peptide comprises at least about 1, 2, 3, 5, 10, 20, or 30 amino acid residues from amino acid residues 1- 763 of SEQ ID NO: 8. DNA and polypeptide analogues and fragments of analogues and original DNA and polypeptide CR4 sequences are also pan of the invention, in the form described above for CRI. The sequence of this gene is identical to a gene reported as SATB-1 (Dickinson, L. A. et al., Cell 70: 631-645 (1992)), for Special AT-rich Binding protein 1, which binds seleαively to the nuclear matrix/scaffold-associating region of DNA. It is expressed exclusively in the thymus and aαivated peripheral T cells. CR4 is the only CR gene also aαivated by the TCR. In addition, elevated cAMP actually stimulates CR4 gene expression. Because the CR4 gene produα binds to special AT-rich regions of DNA known to associate with proteins in the nuclear mating, CR4 is most likely a novel nuclear matrix protein. The nuclear matrix proteins are known to influence the structure of DNA, facilitating transcription of specific genes in particular differentiated tissues. Because the expression of CR4 is restriαed to thymocytes and aαivated T cells, it is likely that CR4 plays an important role in T cell maturation, differentiation or proliferation. Therefore, novel agents that modify CR4 gene expression or CR4 funαion have the potential to be useful to manipulate the T cell immune response. Thus, CR4 can be used in an assay to identify such novel agents which can be used, for example, to treat transplant recipients by, for example, inhibiting the recipient's T cell immune response. These agents can also be used to stimulate the T cell immune response in immunosuppressed subjects, e.g., AIDS patients. The CR5 gene comprises a 2020 nucleotide fragment of SEQ. ID NO: 9, which encodes a protein of 258 amino acids (about 28 kD), of SEQ. ID NO: 10. The sequences are shown in Table VI below.
Table VI: Full DNA and Deduced Protein Sequence for CR5
CGCCCGCGCG CCCCGGGAGC CTACCCAGCA CGCGCTCCGC GCCCACTGGT TCCCTCCAGC 60 CGCCGCCGTC CAGCCGAGTC CCCACTCCGG AGTCGCCGCT GCCGCGGGGA C ATG GTC 117
Met Val 1 CTC TGC GTT CAG GGA CCT CGT CCT TTG CTG GCT GTG GAG CGG ACT GGG 165 Leu Cys Val Gin Gly Pro Arg Pro Leu Leu Ala Val Glu Arg Thr Gly 5 10 15
CAG CGG CCC CTG TGG GCC CCG TCC CTG GAA CTG CCC AAG CCA GTC ATG 213 Gin Arg Pro Leu Trp Ala Pro Ser Leu Glu Leu Pro Lys Pro Val Met 20 25 30 CAG CCC TTG CCT GCT GGG GCC TTC CTC GAG GAG GTG GCA GAG GGT ACC 261 Gin Pro Leu Pro Ala Gly Ala Phe Leu Glu Glu Val Ala Glu Gly Thr 35 40 45 50 Table VI: Full DNA and Deduced Protein Sequence for CR5 (Cont'd)
CCA GCC CAG ACA GAG AGT GAG CCA AAG GTG"CTG GAC CCA GAG GAG GAT 309 Pro Ala Gin Thr Glu Ser Glu Pro Lys Val Leu Asp Pro Glu Glu Asp 55 ' 60 65
CTG CTG TGC ATA GCC AAG ACC TTC TCC TAC CTT CGG GAA TCT GGC TGG 357 Leu Leu Cys He Ala Lys Thr Phe^Ser Tyr Leu Arg Glu Ser Gly Trp 70 ^ 75 80 TAT TGG GGT TCC ATT ACG GCC AGC GAG GCC CGA CAA CAC CTG CAG AAG 405 Tyr Trp Gly Ser He Thr Ala Ser Glu Ala Arg Gin His Leu Gin Lys
85 90 95
ATG CCA GAA GGC ACG TTC TTA GTA CGT GAC AGC ACG CAC CCC AGC TAC 453 Met Pro Glu Gly Thr Phe Leu Val Arg Asp Ser Thr His Pro Ser Tyr 100 105 110
CTG TTC ACG CTG TCA GTG AAA ACC ACT CGT GGC CCC ACC AAT GTA CGC 501
Leu Phe Thr Leu Ser Val Lys Thr Thr Arg Gly Pro Thr Asn Val Arg
115 120 125 130
ATT GAG TAT GCC GAC TCC AGC TTC CGT CTG GAC TCC AAC TGC TTG TCC 549 He Glu Tyr Ala Asp Ser Ser Phe Arg Leu Asp Ser Asn Cys Leu Ser
135 140 145
AGG CCA CGC ATC CTG GCC TTT CCG GAT GTG GTC AGC CTT GTG CAG CAC 597 Arg Pro Arg He Leu Ala Phe Pro Asp Val Val Ser Leu Val Gin His 150 155 160 TAT GTG GCC TCC TGC ACT GCT GAT ACC CGA AGC GAC AGC CCC GAT CCT 645 Tyr Val Ala Ser Cys Thr Ala Asp Thr Arg Ser Asp Ser Pro Asp Pro
165 170 175
GCT CCC ACC CCG GCC CTG CCT ATG CCT AAG GAG GAT GCG CCT AGT GAC 693 Ala Pro Thr Pro Ala Leu Pro Met Pro Lys Glu Asp Ala Pro Ser Asp 180 185 190
CCA GCA CTG CCT GCT CCT CCA CCA GCC ACT GCT GTA CAC CTA AAA CTG 741
Pro Ala Leu Pro Ala Pro Pro Pro Ala Thr Ala Val His Leu Lys Leu
195 200 205 210
GTG CAG CCC TTT GTA CGC AGA AGA AGT GCC CGC AGC CTG CAA CAC CTG 789 Val Gin Pro Phe Val Arg Arg Arg Ser Ala Arg Ser Leu Gin His Leu
215 220 225
TGC CGC CTT GTC ATC AAC CGT CTG GTG GCC GAC GTG GAC TGC CTG CCA 837 Cys Arg Leu Val He Asn Arg Leu Val Ala"Asp Val Asp Cys Leu Pro 230 235 240 CTG CCC CGG CGC ATG GCC GAC TAC CTC CGA CAG TAC CCC TTC CAG CTC T 886 Leu Pro Arg Arg Met Ala Asp Tyr Leu Arg Gin Tyr Pro Phe Gin Leu 245 250 255
(SEQ. ID No: 10) GACTGTACGG GGCAATCTGC CCACCCTCAC CCAGTCGCAC CCTGGAGGGG ACATCAGCCC 946 CAGCTGGACT TGGGCCCCCA CTGTCCCTCC TCCAGGCATC CTGGTGCCTG CATACCTCTG 1006 GCAGCTGGCC CAGGAAGAGC CAGCAAGAGC AAGGCATGGG AGAGGGGAGG TGTCACACAA 1066 CTTGGAGGTA AATGCCCCCA GGCCGCATGT GGCTTCATTA TACTGAGCCA TGTGTCAGAG 1126 GATGGGGAGA CAGGCAGGAC CTTGTCTCAC CTGTGGGCTG GGCCCAGACC TCCACTCGCT 1186 TGCCTGCCCT GGCCACCTGA ACTGTATGGG CACTCTCAGC CCTGGTTTTT CAATCCCCAG 1246 GGTCGGGTAG GACCCCTACT GGCAGCCAGC CTCTGTTTCT GGGAGGATGA CATGCAGAGG 1306 AACTGAGATC GACAGTGACT AGTGACCCCT TGTTGAGGGG TAAGCCAGGC TAGGGGACTG 1366 CACAATTATA CACTCCTGAG CCCTGGTAGT CCAGAGACCC CAACTCTGCC CTGGCTTCTC 1426 TGGTTCTTCC CTGTGGAAAG CCCATCCTGA GACATCTTGC TGGAACCAAG GCAATCCTGG 1486 ATGTCCTGGT ACTGACCCAC CCGTCTGTGA ATGTGTCCAC TCTCTTCTGC CCCCAGCCAT 1546 ATTTGGGGAG GATGGACAAC TACAATAGGT AAGAAAATGC AGCCGGAGCC TCAGTCCCCA 1606 Table VI: Full DNA and Deduced Protein Sequence for CR5 (Cont'd)
GCAGAGCCTG TGTCTCACCC CCTCACAGGA CAGAGCTGTA TCTGCATAGA GCTGGTCTCA 1666
CTGTGGCGCA GGCCCCGGGG GGAGTGCCTG TGCTGTCAGG AAGAGGGGGT GCTGGTTTGA 1726
GGGCCACCAC TGCAGTTCTG CTAGGTCTGC TTCCTGCCCA GGAAGGTGCC TGCACATGAG 1786
AGGAGAGAAA TACACGTCTG ATAAGACTTC ATGAAATAAT AATTATAGCA AAGAACAGTT 1846
TGGTGGTCTT TTCTCTTCCA CTGATTTTTC TGTAATGACA TTATACCTTT ATTACCTCTT 1906
TATTTTATTA CCTCTATAAT AAAATGATAC CTTTCATGTA AAAAAAAAAA AAAA 1960
(SEQ. ID"No: 9)
Recent studies have identified another gene product, termed VHL for Von Hippel Lindau Disease, that also binds to the p 15 subunit. The VHL gene is thought to encode a novel tumor suppressor gene, in which persons with mutant VHL genes have a high incidence of tumors, particularly renal cell carcinomas, and pheochromocytomas. Since CR5 expression is markedly induced during IL-2-promoted T-cell proliferation, it is likely that CR5 potentiates mRNA transcript elongation, thereby antagonizing the suppressive effect of VHL.
Another aspect of the present invention pertains to a CR5 polypeptide, preferably a substantially pure preparation of a CR5 polypeptide, or a recombinant CR5 polypeptide. In preferred embodiments, the CR5 polypeptide comprises an amino acid sequence at least about 60% to 95%, preferably about 80% to 90% homologous to the amino acid sequence in SEQ ID NO: 10, essentially the same as the SEQ ID NO: 10, at least about 5 to 150, preferably about 10 to 100, more preferably about 210 to 50 amino acids in length, comprises at least about 5 to 150, preferably at least about 10 to 100, more preferably at least about 20 to 50, contiguous amino acids from SEQ ID NO: 10. In further preferred embodiments, a protein homologous to SEQ ID NO: 10 has a molecular weight in the range of 20-35 kD, preferably about 28 kD. In a preferred embodiment, a polypeptide having at least one biological activity of the CR5 polypeptide may differ in amino acid sequence from the SEQ ID NO: 10, but such differences result in a modified polypeptide which functions in the same or similar manner, or has the same or similar characteristics as native CR5 protein. Such a peptide can include at least about 1, 2, 3, or 5, and preferably about 10, 20, and 30, amino acid residues from residues 1-258 of SEQ ID NO: 10.
In yet other preferred embodiments, the CR5 polypeptide is a recombinant fusion protein which comprises a second polypeptide portion, e.g., having an amino acid sequence unrelated to a protein represented by SEQ ED NO: 10, e.g., glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain, e.g., the fusion protein is functional in a two-hybrid assay. Yet another aspect of the present invention concerns an immunogen comprising a CR5 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the CR5 polypeptide; e.g. a humoral, antibody, or cellular response. In preferred embodiments, the immunogen comprises an antigenic determinant, e.g. a unique determinant, from a protein represented by SEQ ID NO: 10. A further aspect of the present invention features a monoclonal or polyclonal antibody preparation specifically reactive with an epitope of the CR5 immunogen. Another aspect of the present invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes a CR5 polypeptide, analogues and/or fragments thereof. In certain preferred embodiments, the CR5 nucleic acid comprises a transcriptional regulatory sequence, e.g., at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the CR5 gene sequence, e.g., to render the CR5 gene sequence suitable for use as an expression vector. In yet a further preferred embodiment, the nucleic acid which encodes an CR5 polypeptide of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least about 12 to 150, more preferably to at least about 20 to 100, and still more preferably to at least about 40 to 60 consecutive nucleotides of SEQ ID NO: 9. In yet a further preferred embodiment, the CR5 encoding nucleic acid hybridizes to a nucleic acid probe corresponding to a subsequence encoding at least 4 to 60 consecutive amino acids, more preferably at least about 10 to 50, and even more preferably at least about 20 to 30 amino acid residues between residues 1-258 of SEQ ID NO: 10. In preferred embodiments, the nucleic acid sequence comprises at least about 1, 2, 3 or 5, and preferably at least about 10, 20, 50, or 100 nucleotides from the region of SEQ ID NO: 9, which encodes amino acid residues 1-258 of SEQ ID NO: 10. The encoded peptide comprises at least about 1, 2, 3, 5, 10, 20, or 30 amino acid residues from amino acid residues 1-258 of SEQ ID NO: 10. DNA and polypeptide analogues and fragments of analogues and original DNA and polypeptide CR5 sequences are also part of the invention, in the form described above for CRI .
In the middle of the open reading frame of the CR5 protein is about a 100 amino acid region that has sequence homology (about 25-35%) to src homology 2 (SH2) domains (Waksman, G. et al. (1993) Cell 72: 779-790), found in many proteins that bind to phosphotyrosine residues, e.g., kinases, substrates, linking molecules, and transcription faαors. On either side of this SH2 domain the amino acid sequence is very rich in proline residues. Analysis of CR5 protein expression by different tissues reveals a high level of expression in heart, placenta, lung, liver skeletal muscle and kidney. CR5 protein expression is induced by the proliferation-promoting cytokines IL-2, IL-3, IL-4, IL-5, but not by IL-6. Also, CR5 protein expression is induced by IFN-β and elevated intracellular cAMP, both of which antagonize IL-2 promoted proliferation. CR5 protein has been found to interaα with a subunit of the RNA polymerase II preinitiation complex, termed RNA polymerase II elongation factor SHI, pl5 subunit (Garret, K.P. et al., P.N.A.S. (USA) 91: 5237-5241 (1994)). The pl5 subunit of this RNA polymerase II elongation faαor is known to be responsible for promoting the elongation of transcripted mRNA molecules. Therefore, CR5 appears to funαion as a ligand-stimulated faαor that facilitates mRNA expression by promoting the full elongation of mRNA transcripts. This phenomenon promises to be a novel way in which ligand-receptor systems can regularly promote gene expression. Previously, attention has focused almost entirely on this initiation of transcription, not the elongation of transcripts that were prematurely truncated. Accordingly, novel agents or drugs that modify CR5 gene expression or CR5 function have the potential to provide new ways to alter ligand-stimulated gene expression and thereby alter cellular function.
The CR6 gene includes a 1066 nucleotide fragment of SEQ. ID NO: 11, which encodes protein of 159 amino acids (about 17.5 kD) of SEQ. ID NO: 12. These sequences are shown in Table VII below.
Table VII: Full DNA and Deduced Protein Sequence for CR6
GTGGGTGCGC CGTGCTGAGC TCTGGCTGTC AGTGTGTTCG CCCGCGTCCC CTCCGCGCTC 60 TCCGCTTGTG GATAACTAGC TGCTGGTTGA TCGCACT ATG ACT CTG GAA GAA GTC 115
Met Thr Leu Glu Glu Val 1 5 CGC GGC CAG GAC ACA GTT CCG GAA AGC ACA GCC AGG ATG CAG GGT GCC 163 Arg Gly Gin Asp Thr Val Pro Glu Ser Thr Ala Arg Met Gin Gly Ala
10 15 20
GGG AAA GCG CTG CAT GAG TTG CTG CTG TCG GCG CAG CGT CAG GGC TGC 211 Gly Lys Ala Leu His Glu Leu Leu Leu Ser Ala Gin Arg Gin Gly Cys 25 30 35
CTC ACT GCC GGC GTC TAC GAG TCA GCC AAA GTC TTG AAC GTG GAC CCC 259 Leu Thr Ala Gly Val Tyr Glu Ser Ala Lys Val Leu Asn Val Asp Pro
40 - 45 50
GAC AAT GTG ACC TTC TGT GTG CTG GCT GCG GGT GAG GAG GAC GAG GGC 307 Asp Asn Val Thr Phe Cys Val Leu Ala Ala Gly Glu Glu Asp Glu Gly 55 60 65 70
GAC ATC GCG CTG CAG ATC CAT TTT ACG CTG ATC CAG GCT TTC TGC TGC 355 Asp He Ala Leu Gin He His Phe Thr Leu He Gin Ala Phe Cys Cys 75 80 85 GAG AAC GAC ATC GAC ATA GTG CGC GTG GGC GAT GTG CAG CGG CTG GCG 403 Glu Asn Asp He Asp He Val Arg Val Gly Asp Val Gin Arg Leu Ala
90 95 100
GCT ATC GTG GGC GCC GGC GAG GAG GCG GGT GCG'CCG GGC GAC CTG CAC 451 Ala He Val Gly Ala Gly Glu Glu Ala Gly Ala Pro Gly Asp Leu His 105 110 115
TGC ATC CTC ATT TCG AAC CCC AAC GAG GAC GCC TGG AAG GAT CCC GCC 499 Cys He Leu He Ser Asn Pro Asn Glu Asp Ala Trp Lys Asp Pro Ala
120 125 130
TTG GAG AAG CTC AGC CTG TTT TGC GAG GAG AGC CGC AGC GTT AAC GAC 547 Leu Glu Lys Leu Ser Leu Phe Cys Glu Glu Ser Arg Ser Val Asn Asp 135 140 145 150 Table VII: Full DNA and Deduced Protein Sequence for CR6
TGG GTG CCC AGC ATC ACC CTC CCC GAG T GACAGCCCGG CGGGGACCTT 595 Trp Val Pro Ser He Thr Leu Pro Glu (SEQ. ID No: 12) 155
GGTCTGATCG ACGTGGTGAC GCCCCGGGGC GCCTAGAGCG CGGCTGGCTC TGTGGAGGGG 655
CCCTCCGAGG GTGCCCGAGT GCGGCGTGGA GACTGGCAGG CGGGGGGGGC GCCTGGAGAG 715
CGAGGAGGCG CGGCCTCCCG AGGAGGGGCC CGGTGGCGGC AGGGCCAGGC TGGTCCGAGC 775
TGAGGACTCT GCAAGTGTCT GGAGCGGCTG CTCGCCCAGG AAGGCCTAGG CTAGGACGTT 835 GGCCTCAGGG CCAGGAAGGA CAGACTGGCC GGGCAGGCGT GACTCAGCAG CCTGCGCTCG 895
GCAGGAAGGA GCGGCGCCCT GGACTTGGTA CAGTTTCAGG AGCGTGAAGG ACTTAACCGA 955
CTGCCGCTGC TTTTTCAAAA CGGATCCGGG CAATGCTTCG TTTTCTAAAG GATGCTGCTG 1015
TTGAGCTTTG AATTTTACAA TAAACTTTTT GAAACAAAAA AAAAAAAAAA 1065
(SEQ. ID No: 11)
The present invention further pertains to a CR6 polypeptide, preferably a substantially pure preparation of a CR6 polypeptide, or a recombinant CR6 polypeptide. In preferred embodiments, the CR6 polypeptide comprises an amino acid sequence at least about 60% to 95%, preferably about 80% to 90%, homologous to the amino acid sequence in SEQ ID NO: 12, essentially the same as the amino acid sequence in SEQ ID NO: 12, comprises at least about 5 to 150, preferably about 10 to 100, more preferably about 20 to 50 amino acids in length, at least about 5 to 150, preferably at least about 10 to 100, more preferably at least about 20 to 50, more preferably at least about 50, 100, or 150 contiguous amino acids from SEQ ID NO: 12. In further preferred embodiments, a protein homologous to SEQ ID NO: 12 has a molecular weight in the range of 15-25 kD, preferably about 17 kD. In a preferred embodiment, a polypeptide having at least one biological activity of the CR6 polypeptide may differ from the sequence in SEQ ID NO: 12, but such differences result in a modified polypeptide which functions in the same or similar manner as, or which has the same or similar characteristics as, native CR6 protein. Such a peptide can comprise at least about 1 to 100, 2, 3, or 5 to 80, and preferably about 10 to 70, 20, and 30 to 50, amino acid residues from residues 1-159 of SEQ ID NO: 12. In yet other preferred embodiments, the CR6 polypeptide is a recombinant fusion protein which comprises a second polypeptide portion, e.g., having an amino acid sequence unrelated to a protein represented by SEQ ID NO: 12, e.g., the second polypeptide portion is glutathione-S-transferase, a DNA binding domain, a polymerase activating domain, e.g. the fusion protein is functional in a two-hybrid assay. Yet another aspect of the present invention concerns an immunogen comprising a CR6 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the CR6 polypeptide; e.g. a humoral, an antibody, or a cellular response. In preferred embodiments, the immunogen comprises an antigenic determinant, e.g. a unique determinant, from a protein represented by SEQ ID NO: 12. A further aspect of the present invention features a monoclonal or polyclonal antibody preparation specifically reactive with an epitope of the CR6 immunogen.
Another aspect of the present invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes a CR6 polypeptide, analogues and/or fragments thereof. In certain preferred embodiments, the CR6 nucleic acid comprises a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the CR6 gene sequence, e.g., to render the CR6 gene sequence suitable for use as an expression vector. In yet a further preferred embodiment, the nucleic acid which encodes an CR6 polypeptide of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least about 12 and more preferably to at least about 20, and more preferably to at least about 40 consecutive nucleotides of SEQ ID NO: 11. In yet a further preferred embodiment, the CR6 encoding nucleic acid hybridizes to a nucleic acid probe corresponding to a subsequence encoding at least 4 consecutive amino acids, more preferably at least about 10, and even more preferably at least about 20 amino acid residues between residues 1-159 of SEQ ID NO: 12. In preferred embodiments the nucleic acid sequence comprises at least about 1, 2, 3 or 5, and preferably at least about 10, 20, 50, or 100 to 130 nucleotides from the region of SEQ ID NO: 11 which encodes amino acid residues 1-159 of SEQ ID NO: 12. The encoded peptide includes at least about 1, 2, 3, 5, 10, 20, or 30 to 150 amino acid residues from amino acid residues 1-159 of SEQ ID NO: 12. DNA and polypeptide analogues and fragments of analogues and original DNA and polypeptide CR6 sequences are also part of the invention, in the form described above for CRI.
Recent experiments have indicated that GADD45 binds to p21, which is another p53- induced gene. p21 inhibits progression through Gl of the cell cycle by binding to and suppressing cyclin-CDK complexes. In addition, p21 has also been shown to bind to PCNA, preventing DNA replication. It is likely that CR6 also binds to p21, and it may facilitate cellular proliferation, by preventing the inhibitory aαivity of p21.
This gene belongs to a family of small nuclear-localizing gene produαs. Two other members of this family, GADD45 and MyD118, have been identified. GADD45 was cloned from human fibroblasts induced by UV irradiation (Papathanasiou, M. A. et al., Moi. Cell Biol. 11(2): 1009-1016 (1991)). This protein is regulated by p53 and suppresses growth of cells by binding to PCNA, a co-factor required for DNA polymerase δ aαivity. (Smith, M. L. et al., Science 266: 1376-1380 (1994)). MyD118 was cloned from M1D+ myeloid precursors following induαion of terminal differentiation and growth aπest by IL6. (Abdollahi, A. et al., Oncogene 6: 165-167 (1991)). At the nucleotide level, CR6 is about 65% homologous to GADD45. At the protein level, CR6 is about 54% homologous to GADD45. At the nucleotide level, CR6 is about 66% homologous to MyD118. At the protein level, CR6 is about 53% homologous to MyD118. The CR6 protein is expressed only in testes, ovary and prostate, and its expression is suppressed by elevated cAMP. By analogy to it's homology to GADD45 and MyD118, the CR6 gene produα most likely plays a role in DNA replication. Thus far, experiments have indicated that CR6 expression is not induced by agents that damage DNA, such as UV light. Moreover, CR6 does not bind to PCNA. However, CR6 does promote DNA replication in vitro, and it is likely to be a novel CD-factor necessary for DNA replication. Therefore, the CR6 gene product can be used to identify inhibitors of DNA replication which can be used as anti-proliferative agents, e.g., in the treatment of cancer .
The CR7 gene includes a 2400 nucleotide DNA (SEQ. ID No: 27), encoding a protein of 313 amino acids (about 34 kD) of SEQ. ID No: 28. The nucleotide (SEQ. ID No: 27), and amino acid sequence (SEQ. ID No: 28) are shown in Table VII below.
Table VII: Full DNA Sequence and Protein Sequence for CR7
GCGCCGCATC CTGGAGGTTG GG ATG CTC TTG TCC AAA ATC AAC TCG CTT GCC 52 CGCGGCGTAG GACCTCCAAC CC TAC GAG AAC AGG TTT TAG TTG AGC GAA CGG
Met Leu Leu Ser Lys He Asn Ser Leu Ala 1 5 10 CAC CTG CGC GCC CGC GCC TGC AAC GAC CTG CAC GCC ACC AAG CTG GCG 100 GTG GAC GCG CGG GCG CGG ACG TTG CTG GAC GTG CGG TGG TTC GAC CGC His Leu Arg Ala Arg Ala Cys Asn Asp Leu His Ala Thr Lys Leu Ala
15 20 25
CCG GGC AAG GAG AAG GAG CCC CTG GAG TCG CAG TAC CAG GTG GGC CCG 148 GGC CCG TTC CTC TTC CTC GGG GAC CTC AGC GTC ATG GTC CAC CCG GGC Pro Gly Lys Glu Lys Glu Pro Leu Glu Ser Gin Tyr Gin Val Gly Pro
30 35 40
CTA CTG GGC AGC GGC GGC TTC GGC TCG GTC TAC TCA GGC ATC CGC GTC 196 GAT GAC CCG TCG CCG CCG AAG CCG AGC CAG ATG AGT CCG TAG GCG CAG Leu Leu Gly Ser Gly Gly Phe Gly Ser Val Tyr Ser Gly He Arg Val 45 50 55
TCC GAC AAC TTG CCG GTG GCC ATC AAA CAC GTG GAG AAG GAC CGG ATT 244 AGG CTG TTG AAC GGC CAC CGG TAG TTT GTG CAC CTC TTC CTG GCC TAA Ser Asp Asn Leu Pro Val Ala He Lys His Val Glu Lys Asp Arg He 60 65 70
TCC GAC TGG GGA GAG CTG CCT AAT GGC ACT CGA GTG CCC ATG GAA GTG 292 AGG CTG ACC CCT CTC GAC GGA TTA CCG TGA GCT CAC GGG TAC CTT CAC Ser Asp Trp Gly Glu Leu Pro Asn Gly Thr Arg Val Pro Met Glu Val 75 80 85 90 GTC CTG CTG AAG AAG GTG AGC TCG GGT TTC TCC GGC GTC ATT AGG CTC 340 CAG GAC GAC TTC TTC CAC TCG AGC CCA AAG AGG CCG CAG TAA TCC GAG Val Leu Leu Lys Lys Val Ser Ser Gly Phe Ser Gly Val He Arg Leu
95 100 105
CTG GAC TGG TTC GAG AGG CCC GAC AGT TTC GTC CTG ATC CTG GAG AGG 388 GAC CTG ACC AAG CTC TCC GGG CTG TCA AAG CAG GAC TAG GAC CTC TCC Leu Asp Trp Phe Glu Arg Pro Asp Ser Phe Val Leu He Leu Glu Arg
110 115 120
CCC GAG CCG GTG CAA GAT CTC TTC GAC TTC ATC ACG GAA AGG GGA GCC 436 GGG CTC GGC CAC GTT CTA GAG AAG CTG AAG TAG TGC CTT TCC CCT CGG Pro Glu Pro Val Gin Asp Leu Phe Asp Phe He Thr Glu Arg Gly Ala 125 130 135
Table VII: Full DNA Sequence and Protein Sequence for CR7 (Cont'd)
CTG CAA GAG GAG CTG GCC CGC AGC TTC TTC TGG CAG GTG CTG GAG GCC 484 GAC GTT CTC CTC GAC CGG GCG TCG AAG AAG ACC GTC CAC GAC CTC CGG Leu Gin Glu Glu Leu Ala Arg Ser Phe Phe Trp Gin Val Leu Glu Ala
140 145 150
GTG CGG CAC TGC CAC AAC TGC GGG GTG CTC CAC CGC GAC ATC AAG GAC 532 CAC GCC GTG ACG GTG TTG ACG CCC CAC GAG GTG GCG CTG TAG TTC CTG Val Arg His Cys His Asn Cys Gly Val Leu His Arg Asp He Lys Asp 155 160 165 170
GAA AAC ATC CTT ATC GAC CTC AAT CGC GGC GAG CTC AAG CTC ATC GAC 580 CTT TTG TAG GAA TAG CTG GAG TTA GCG CCG CTC GAG TTC GAG TAG CTG Glu Asn He Leu He Asp Leu Asn Arg Gly Glu Leu Lys Leu He Asp
175 180 185
TTC GGG TCG GGG GCG CTG CTC AAG GAC ACC GTC TAC ACG GAC TTC GAT 620 AAG CCC AGC CCC CGC GAC GAG TTC CTG TGG CAG ATG TGC CTG AAG CTA Phe Gly Ser Gly Ala Leu Leu Lys Asp Thr Val Tyr Thr Asp Phe Asp 190 195 200
GGG ACC CGA GTG TAT AGC CCT CCA GAG TGG ATC CGC TAC CAT CGC TAC 668 CCC TGG GCT CAC ATA TCG GGA GGT CTC ACC TAG GCG ATG GTA GCG ATG Gly Thr Arg Val Tyr Ser Pro Pro Glu Trp He Arg Tyr His Arg Tyr 205 210 215 CAT GGC AGG TCG GCG GCA GTC TGG TCC CTG GGG ATC CTG CTG TAT GAT 716 GTA CCG TCC AGC CGC CGT CAG ACC AGG GAC CCC TAG GAC GAC ATA CTA His Gly Arg Ser Ala Ala Val Trp Ser Leu Gly He Leu Leu Tyr Asp
220 225 230
ATG GTG TGT GGA GAT ATT CCT TTC GAG CAT GAC GAA GAG ATC ATC AGG 764 TAC CAC ACA CCT CTA TAA GGA AAG CTC GTA CTG CTT CTC TAG TAG TCC Met Val Cys Gly Asp He Pro Phe Glu His Asp Glu Glu He He Arg 235 240 245 250
GGC CAG GTT TTC TTC AGG CAG AGG GTC TCT TCA GAA TGT CAG CAT CTC 812 CCG GTC CAA AAG AAG TCC GTC TCC CAG AGA AGT CTT ACA GTC GTA GAG Gly Gin Val Phe Phe Arg Gin Arg Val Ser Ser Glu Cys Gin His Leu
255 260 265
ATT AGA TGG TGC TTG GCC CTG AGA CCA TCA GAT AGG CCA ACC TTC GAA 860 TAA TCT ACC ACG AAC CGG GAC TCT GGT AGT CTA TCC GGT TGG AAG CTT He Arg Trp Cys Leu Ala Leu Arg Pro Ser Asp Arg Pro Thr Phe Glu 270 275 280
GAA ATC CAG AAC CAT CCA TGG ATG CAA GAT GTT CTC CTG CCC CAG GAA 908 CTT TAG GTC TTG GTA GGT ACC TAC GTT CTA CAA GAG GAC GGG GTC CTT Glu He Gin Asn His Pro Trp Met Gin Asp Val Leu Leu Pro Gin Glu 285 290 295 ACT GCT GAG ATC CAC CTC CAC AGC CTG TCG CCG GGG CCC AGC AA 952
TGA CGA CTC TAG GTG GAG GTG TCG GAC AGC GGC CCC GGG TCG TT Thr Ala Glu He His Leu His Ser Leu Ser Pro Gly Pro Ser Lys 300 305 310 313 (SEQ. ID No: 27)
ATAGCAGCCT TTCTGGCAGG TCCTCCCCTC TCTTGTCAGA TGCCCAGGAG GGAAGCTTCT 1020 TATCGTCGGA AAGACCGTCC AGGAGGGGAG AGAACAGTCT ACGGGTCCTC CCTTCGAAGA GTCTCCAGCT TTCCCGAGTA CCAGTGACAC GTCTCGCCAA GCAGGACAGT GCTTGATACA 1080 CAGAGGTCGA AAGGGCTCAT GGTCACTGTG CAGAGCGGTT CGTCCTGTCA CGAACTATGT
GGAACAACAT TTACAACTCA TTCCAGATCC CAGGCCCCTG GAGGCTGCCT CCCAACAGTG 1140 CCTTGTTGTA AATGTTGAGT AAGGTCTAGG GTCCGGGGAC CTCCGACGGA GGGTTGTCAC GGGAAGAGTG ACTCTCCAGG GGTCCTAGGC CTCAACTCCT CCCATAGATA CTCTCTTCTT 1200 CCCTTCTCAC TGAGAGGTCC CCAGGATCCG GAGTTGAGGA GGGTATCTAT GAGAGAAGAA CTCATAGGTG TCCAGCATTG CTGGACTCTG AAATATCCCG GGGGTGGGGG GTGGGGGTGG 1260 GAGTATCCAC AGGTCGTAAC GACCTGAGAC TTTATAGGGC CCCCACCCCC CACCCCCACC GTCAGAACCC TGCCATGGAA CTGTTTCCTT CATCATGAGT TCTGCTGAAT GCCGCGATGG 1320 CAGTCTTGGG ACGGTACCTT GACAAAGGAA GTAGTACTCA AGACGACTTA CGGCGCTACC GTCAGGTAGG GGGGAAACAG GTTGGGATGG GATAGGACTA GCACCATTTT AAGTCCCTGT 1380 CAGTCCATCC CCCCTTTGTC CAACCCTACC CTATCCTGAT CGTGGTAAAA TTCAGGGACA
Table VII: Full DNA Sequence and Protein Sequence for CR7 (Cont'd)
CACCTCTTCC GACTCTTTCT GAGTGCCTTC TGTGGGGACT CCGGCTGTGC TGGGAGAAAT 1440 GTGGAGAAGG CTGAGAAAGA CTCACGGAAG ACACCCCTGA GGCCGACACG ACCCTCTTTA
ACTTGAACTT GCCTCTTTTA CCTGCTGCTT CTCCAAAAAT CTGCCTGGGT TTTGTTCCCT 1500 TGAACTTGAA CGGAGAAAAT GGACGACGAA GAGGTTTTTA GACGGACCCA AAACAAGGGA ATTTTTCTCT CCTGTCCTCC CTCACCCCCT CCTTCATATG AAAGGTGCCA TGGAAGAGGC 1560 TAAAAAGAGA GGACAGGAGG GAGTGGGGGA GGAAGTATAC TTTCCACGGT ACCTTCTCCG TACAGGGCCA AACGCTGAGC CACCTGCCCT TTTTTCTCCT CCTTTAGTAA AACTCCGAGT 1620 ATGTCCCGGT TTGCGACTCG GTGGACGGGA AAAAAGAGGA GGAAATCATT TTGAGGCTCA GAACTGGTCT TCCTTTTTGG TTTTTACTTA ACTGTTTCAA AGCCAAGACC TCACACACAC 1680 CTTGACCAGA AGGAAAAACC AAAAATGAAT TGACAAAGTT TCGGTTCTGG AGTGTGTGTG AAAAAATGCA CAAACAATGC AATCAACAGA AAAGCTGTAA ATGTGTGTAC AGTTGGCATG 1740 TTTTTTACGT GTTTGTTACG TTAGTTGTCT TTTCGACATT TACACACATG TCAACCGTAC
GTAGTATACA AAAAGATTGT AGTGGATCTA ATTTTTAAGA AATTTTGCCT TTAAGTTATT 1800 CATCATATGT TTTTCTAACA TCACCTAGAT TAAAAATTCT TTAAAACGGA AATTCAATAA TTACCTGTTT TTGTTTCTTG TTTTGAAAGA TGCGCATTCT AACCTGGAGG TCAATGTTAT 1860 AATGGACAAA AACAAAGAAC AAAACTTTCT ACGCGTAAGA TTGGACCTCC AGTTACAATA GTATTTATTT ATTTATTTAT TTGGTTCCCT TCCTATTCCA AGCTTCGCTG CTGCCCTAGT 1920 CATAAATAAA TAAATAAATA AACCAAGGGA AGGATAAGGT TCGAAGCGAC GACGGGATCA TTTCTTTCCT CCTTTCCTCC TCTGACTTGG GGACCTTTTG GGGGAGGGCT GCGACGCTTG 1980 AAAGAAAGGA GGAAAGGAGG AGACTGAACC CCTGGAAAAC CCCCTCCCGA CGCTGCGAAC CTCTGTTTGT GGGGTGACGG GACTCAGGCG GGACAGTGCT GCAGCTCCCT GGCTTCTGTG 2040 GAGACAAACA CCCCACTGCC CTGAGTCCGC CCTGTCACGA CGTCGAGGGA CCGAAGACAC
GGGCCCCTCA CCTACTTACC CAGGTGGGTC CCGGCTCTGT GGGTGATGGG GAGGGGCATT 2100 CCCGGGGAGT GGATGAATGG GTCCACCCAG GGCCGAGACA CCCACTACCC CTCCCCGTAA GCTGACTGTG TATATAGGAT AATTATGAAA AGCAGTTCTG GATGGTGTGC CTTCCAGATC 2160 CGACTGACAC ATATATCCTA TTAATACTTT TCGTCAAGAC CTACCACACG GAAGGTCTAG CTCTCTGGGG CTGTGTTTTG AGCAGCAGGT AGCCTGCTGG TTTTATCTGA GTGAAATACT 2220 GAGAGACCCC GACACAAAAC TCGTCGTCCA TCGGACGACC AAAATAGACT CACTTTATGA GTACAGGGGA ATAAAAGAGA TCTTATTTTT TTTTTTATAC TTGGCGTTTT TTGAATAAAA 2280 CATGTCCCCT TATTTTCTCT AGAATAAAAA AAAAAATATG AACCGCAAAA AACTTATTTT ACCTTTTGTC TTAAAAC (SEQ. ID No: 25) 2297 TGGAAAACAG AATTTTG (SEQ. ID No: 26) CR7 is an IL-2-induced gene and is a serine/threonine-specific protein kinase involved in T cell lymphomagenesis. The CR7 gene turned out to be identical to the putative proto-oncogene, pim-1, which has been reported to be over-expressed in about 50% of Moloney murine leukemia virus (MuLV)-induced T cell lymphomas (See, Selten, G. et al., Cell 46: 603-611 (1986)). The present invention further pertains to a CR7 polypeptide, preferably a substantially pure preparation of a CR7 polypeptide, or a recombinant CR7 polypeptide. In preferred embodiments, the CR7 polypeptide comprises an amino acid sequence at least about 60% to 95%, preferably about 80% to 90%, homologous to the amino acid sequence in SEQ ID NO: 28; essentially the same as the amino acid sequence in SEQ ID NO: 28, at least about 5 to 150, preferably about 10 to 100, more preferably about 20 to 50 amino acids in length, at least about 5, preferably at least about 10, more preferably at least about 20, more preferably at least about 50, 100, or 150 contiguous amino acids from SEQ ID NO: 28. In further preferred embodiments, a protein homologous to SEQ ID NO: 28 has a molecular weight in the range of about 15-25 kD, preferably about 17 kD. In a preferred embodiment, a polypeptide having at least one biological activity of the CR7 polypeptide may differ in amino acid sequence from the sequence in SEQ ID NO: 28, but such differences result in a modified polypeptide which functions in the same or similar manner as or which has the same or similar characteristics as native CR7 protein. Such a peptide can comprise at least about 1, 2, 3, or 5 to 140, and preferably about 10, 20, and 30 to 100, amino acid residues from residues 1-159 of SEQ ED NO: 28. In yet other preferred embodiments, the CR7 polypeptide is a recombinant fusion protein which comprises a second polypeptide portion, e.g., having an amino acid sequence unrelated to a protein represented by SEQ ID NO: 28, e.g., the second polypeptide portion is glutathione-S-transferase, a DNA binding domain, a polymerase activating domain, e.g. the fusion protein is functional in a two-hybrid assay. Yet another aspect of the present invention concerns an immunogen comprising a CR7 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the CR7 polypeptide; e.g. a humoral response, an antibody response; a cellular response. In preferred embodiments, the immunogen comprises an antigenic determinant, e.g. a unique determinant, from a protein represented by SEQ ID NO: 28. A further aspect of the present invention features a monoclonal or polyclonal antibody preparation specifically reactive with an epitope of the CR7 immunogen.
Another aspect of the present invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes a CR7 polypeptide, analogues and/or fragments thereof. Furthermore, in certain preferred embodiments, the CR7 nucleic acid comprises a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the CR7 gene sequence, e.g., to render the CR7 gene sequence suitable for use as an expression vector. In yet a further prefened embodiment, the nucleic acid which encodes an CR7 polypeptide of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least about 12 to 300, and more preferably to at least about 20 to 200, and more preferably to at least about 40 to 100 consecutive nucleotides of SEQ ID NO: 27. In yet a further preferred embodiment, the CR7 encoding nucleic acid hybridizes to a nucleic acid probe corresponding to a subsequence encoding at least about 4 consecutive amino acids, more preferably at least about 10, and even more preferably at least about 20 amino acid residues between residues 1-159 of SEQ ID NO: 28. In preferred embodiments the nucleic acid sequence comprises at least about 1, 2, 3 or 5, and preferably at least about 10, 20, 50, or 100 to 400 nucleotides from the region of SEQ ID NO: 27 which encodes amino acid residues 1-159 of SEQ ID NO: 28. The encoded peptide includes at least about 1, 2, 3, 5, 10, 20, or 30 to 100 amino acid residues from amino acid residues 1-159 of SEQ ID NO: 28. DNA and polypeptide analogues and fragments of analogues and original DNA and polypeptide CR7 sequences are also part of the invention, in the form described above for CRI.
The CR8 gene includes a 2980 nucleotide fragment of SEQ. ID NO: 13, which encodes (via a 3.2 Kb mRNA transcript) a protein of 412 amino acids (about 45 kD) of SEQ. ID NO: 14. These sequences are shown in Table VIII below.
Table VIII: Full DNA and Deduced Protein Sequence for CR8
CACACCGCCA GTCTGTGCGC TGAGTCGGAG CCAGAGGCCG CGGGGACACC GGGCCATGCA 60 CGCCCCCAAC TGAAGCTGCA TCTCAAAGCC GAAGATTCCA GCAGCCCAGG GGATTTCAAA 120 GAGCTCAGAC TCAGAGGAAC ATCTGCGGAG AGACCCCCGA AGCCCTCTCC AGGGCAGTCC 180 TCATCCAGAC GCTCCGTTAG TGCAGACAGG AGCGCGCAGT GGCCCCGGCT CGCCGCGCC 239 ATG GAG CGG ATC CCC AGC GCG CAA CCA CCC CCC GCC TGC CTG CCC AAA 287 Met Glu Arg He Pro Ser Ala Gin Pro Pro Pro Ala Cys Leu Pro Lys
1 5 10 15
GCA CCG GGA CTG GAG CAC CGA GAC CTA CCA GGG ATG TAC CCT GCC CAC 335 Ala Pro Gly Leu Glu His Arg Asp Leu Pro Gly Met Tyr Pro Ala His
20 25 30
ATG TAC CAA GTG TAC AAG TCA AGA CGG GGA ATA AAG CGG AGC GAG GAC 383 Met Tyr Gin Val Tyr Lys Ser Arg Arg Gly He Lys Arg Ser Glu Asp 35 40 45 AGC AAG GAG ACC TAC AAA TTG CCG CAC CGG CTC TTC GAG AAA AAG AGA 431 Ser Lys Glu Thr Tyr Lys Leu Pro His Arg Leu Phe Glu Lys Lys Arg
50 55 60
CGT GAC CGG ATT AAC GAG TGC ATC GCC CAG CTG AAG GAT CTC CTA CCC 479 Arg Asp Arg He Asn Glu Cys He Ala Gin Leu Lys Asp Leu Leu Pro 65 70 75 80
GAA CAT CTC AAA CTT ACA ACT TTG GGT CAC TTG GAA AAA GCA GTG GTT 527 Glu His Leu Lys Leu Thr Thr Leu Gly His Leu Glu Lys Ala Val Val
85 * 90 95
CTT GAA CTT ACC TTG AAG CAT GTG AAA GCA CTA ACA AAC CTA ATT GAT 575 Leu Glu Leu Thr Leu Lys His Val Lys Ala Leu Thr Asn Leu He Asp 100 105 110
CAG CAG CAG CAG AAA ATC ATT GCC CTG CAG AGT GGT TTA CAA GCT GGT 623
Gin Gin Gin Gin Lys He He Ala Leu Gin Ser Gly Leu Gin Ala Gly 115 120 125
GAG CTG TCA GGG AGA AAT GTC GAA ACA GGT CAA GAG ATG TTC TGC TCA 671
Glu Leu Ser Gly Arg Asn Val Glu Thr Gly Gin Glu Met Phe Cys Ser
130 135 140
GGT TTC CAG ACA TGT GCC CGG GAG GTG CTT CAG TAT CTG GCC AAG CAC 719
Gly Phe Gin Thr Cys Ala Arg Glu Val Leu Gin Tyr Leu Ala Lys His
145 150 155 160
GAG AAC ACT CGG GAC CTG AAG TCT TCG CAG CTT GTC ACC CAC CTC CAC 767
Glu Asn Thr Arg Asp Leu Lys Ser Ser Gin Leu Val Thr His Leu His 165 170 175
CGG GTG GTC TCG GAG CTG CTG CAG GGT GGT ACC TCC AGG AAG CCA TCA 815
Arg Val Val Ser Glu Leu Leu Gin Gly Gly Thr Ser Arg Lys Pro Ser 180 185 190
GAC CCA GCT CCC AAA GTG ATG GAC TTC AAG GAA AAA CCC AGC TCT CCG 863
Asp Pro Ala Pro Lys Val Met Asp Phe Lys Glu Lys Pro Ser Ser Pro 195 200 205
GCC AAA GGT TCG GAA GGT CCT GGG AAA AAC TGC GTG CCA GTC ATC CAG 911
Ala Lys Gly Ser Glu Gly Pro Gly Lys Asn Cys Val Pro Val He Gin 210 215 220
Table VIII: Full DNA and Deduced Protein Sequence for CR8 (Cont'd)
CGG ACT TTC GCT CAC TCG AGT GGG GAG CAG AGC GGC AGC GAC ACG GAC 959 Arg Thr Phe Ala His Ser Ser Gly Glu Gin Ser Gly Ser Asp Thr Asp 225 230 235 240
ACA GAC AGT GGC TAT GGA GGA GAT TCG GAG AAG GGC GAC TTG CGC AGT 1007 Thr Asp Ser Gly Tyr Gly Gly Asp Ser Glu Lys Gly Asp Leu Arg Ser
245 250 255
GAG CAG CCG TGC TTC AAA AGT GAC CAC GGA CGC AGG TTC ACG ATG GGA 1055 Glu Gin Pro Cys Phe Lys Ser Asp His Gly Arg Arg Phe Thr Met Gly 260 265 270
GAA AGG ATC GGC GCA ATT AAG CAA GAG TCC GAA GAA CCC CCC ACA AAA 1103 Glu Arg He Gly Ala He Lys Gin Glu Ser Glu Glu Pro Pro Thr Lys 275 280 285 AAG AAC CGG ATG CAG CTT TCG GAT GAT GAA GGC CAT TTC ACT AGC AGT 1151 Lys Asn Arg Met Gin Leu Ser Asp Asp Glu Gly His Phe Thr Ser Ser
290 295 300
GAC CTG ATC AGC TCC CCG TTC CTG GGC CCA CAC CCA CAC CAG CCT CCT 1199 Asp Leu He Ser Ser Pro Phe Leu Gly Pro His Pro His Gin Pro Pro 305 310 315 320
TTC TGC CTG CCC TTC TAC CTG ATC CCA CCT TCA GCG ACT GCC TAC CTG 1247 Phe Cys Leu Pro Phe Tyr Leu He Pro Pro Ser Ala Thr Ala Tyr Leu
325 330 335
CCC ATG CTG GAG AAG TGC TGG TAT CCC ACC TCA GTG CCA GTG CTA TAC 1295 Pro Met Leu Glu Lys Cys Trp Tyr Pro Thr Ser Val Pro Val Leu Tyr 340 345 350
CCA GGC CTC AAC GCC TCT GCC GCA GCC CTC TCT AGC TTC ATG AAC CCA 1343 Pro Gly Leu Asn Ala Ser Ala Ala Ala Leu Ser Ser Phe Met Asn Pro 355 360 365 GAC AAG ATC TCG GCT CCC TTG CTC ATG CCC CAG AGA CTC CCT TCT CCC 1391 Asp Lys He Ser Ala Pro Leu Leu Met Pro Gin Arg Leu Pro Ser Pro
370 375 380
TTG CCA GCT CAT CCG TCC GTC GAC TCT TCT GTC TTG CTC CAA GCT CTG 1439 Leu Pro Ala His Pro Ser Val Asp Ser Ser Val Leu Leu Gin Ala Leu 385 390 395 400
AAG CCA ATC CCC CCT TTA AAC TTA GAA ACC AAA GAC T AAACTCTCTA 1486
Lys Pro He Pro Pro Leu Asn Leu Glu Thr Lys Asp (SEQ. ID No: 14)
405 410
GGGGATCCTG CTGCTTNGCT TTCCTNCCTC GCTACTTCCT AAAAAGCAAC CNNAAAGNTT 1546 TNGTGAATGC TGNNAGANTG TTGCATTGTG TATACTGAGA TAATCTGAGG CATGGAGAGC 1606 AGANNCAGGG TGTGTGTGTG TGTGTGTGTG TGTGTGTGTG TATGTGCGTG TGCGTGCACA 1666 TGTGTGCCTG CGTGTTGGTA TAGGACTTTA NNGCTCCTTNN GGCATAGGG AAGTCACGAA 1726 GGATTGCTNG ACATCAGGAG ACTNGGGGGG GATTGTAGCA GACGTCTGGG CTTNNCCCCA 1786 CCCAGAGAAT AGCCCCCNNC NANACANATC AGCTGGATTT ACAAAAGCTT CAAAGTCTTG 1846 GTCTGTGAGT CACTCTTCAG TTTGGGAGCT GGGTCTGTGG CTTTGATCAG AAGGTACTTT 1906 CAAAAGAGGG CTTTCCAGGG CTCAGCTCCC AACCAGCTGT TAGGACCCCA CCCTTTTGCC 1966 TTTATTGTCG ACGTGACTCA CCAGACGTCG GGGAGAGAGA GCAGTCAGAC CGAGCTTTTC 2026 TGCTAACATG GGGAGGGTAG CAGACACTGG CATAGCACGG TAGTGGTTTG GGGGAGGGTT 2086 TCCGCAGGTC TGCTCCCCAC CCCTGCCTCG GAAGAATAAA GAGAATGTAG TTCCCTACTC 2146 AGGCTTTCGT AGTGATTAGC TTACTAAGGA ACTGAAAATG GGCCCCTTGT ACAAGCTGAG 2206 CTGCCCCGGA GGGAGGGAGG AGTTCCCTGG GCTTCTGGCA CCTGTTTCTA GGCCTAACCA 2266 TTAGTACTTA CTGTGCAGGG AACCAAACCA AGGTCTGAGA AATGCGGACA NCCCGAGCGA 2326 GCACCCCAAA GTGCACAAAG CTGAGTAAAA AGCTGCCCCC TTCAAACAGA ACTAGACTCA 2386 GTTTTCAATT CCATCCTAAA ACTCCTTTTA ACCAAGCTTA GCTTCTCAAA GGGCTAACCA 2446 AGCCTTGGAA CCGCCAGATC CTTTCTGTAG GCTAATTCCT CTTGGCCAAC GGCATATGGA 2506 GTGTCCTTAT TGCTAAAAAG GATTCCGNCT CCTTCAAAGA AGTTTTATTT TTGGTCCAGA 2566 GTACTTGTTT TCCCGATGTG TCCAGCCAGC TCCGCAGCAG CTTTTCAAAA TGCACTATGC 2626 CTGATTGCTG ATCGTGTTTT AACTTTTTCT TTTCCTGTTT TTATTTTGGT ATTAAGTCGC 2686 TGGCTTTATT TGTAAAGCTG TTATAAATAT ATATTATATN AANTATATTA AAAAGGAAAN 2746 TGTTNCAGAT GTTTATTTGT ATAATTACTT GATTCACANA GNGAGAAAAA NTGANTGTAT 2806 TCCTGTNTTN GAAGAGAAGA NNAATTTTTT TTTTCTCTAG GGAGAGGTAC AGNGTTNNTN 2866
Table VIII: Full DNA and Deduced Protein Sequence for CR8 (Cont'd)
TTTTGGGGCC TNCCNGAAGG GGTAAANNNG AAAATNTTTC TATNTATGAG TAAATGTTAA 2926 GTAGTTGTNT NAAAATACTN AATAAAATAA TTCTCTCCCT GTGGNNGAGA NAAC 2980 (SEQ. ID No: 13)
The present invention still further pertains to a CR8 polypeptide, preferably a substantially pure preparation of a CR8 polypeptide, or a recombinant CR8 polypeptide. In preferred embodiments, the CR8 polypeptide comprises an amino acid sequence at least about 60%), to 95%, preferably about 80% to 90% homologous to the amino acid sequence in SEQ ED NO: 14, essentially the same as the amino acid sequence in SEQ ID NO: 14, comprises about 5 to 150, preferably about 10 to 100, more preferably about 20 to 50 amino acids in length, about 5 to 300, preferably at least about 10 to 200, more preferably at least about 20 to 100, more preferably at least about 50, 100, or 150 to 350 contiguous amino acids from SEQ ID NO: 14. In further preferred embodiments, a protein homologous to SEQ ED NO: 14 has a molecular weight in the range of about 35-50 kD, preferably about 45 kD. In a preferred embodiment, a polypeptide having at least one biological activity of the CR8 polypeptide may differ in amino acid sequence from the sequence in SEQ ID NO: 14, but such differences result in a modified polypeptide which functions in the same or similar manner as or which has the same or similar characteristics as native CR8 protein. Such a peptide can comprise at least about 1, 2, 3, or 5, and preferably about 10, 20, and 30, amino acid residues from residues 1-412 of SEQ ED NO: 14.
In yet other preferred embodiments, the CR8 polypeptide is a recombinant fusion protein which includes a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated to a protein represented by SEQ ED NO: 14, e.g., the second polypeptide portion is glutathione-S-transferase, a DNA binding domain, a polymerase activating domain, e.g., the fusion protein is functional in a two-hybrid assay. Yet another aspect of the present invention pertains to an immunogen comprising a CR8 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for said CR8 polypeptide; e.g. a humoral, antibody, or cellular response. In preferred embodiments, the immunogen comprises an antigenic determinant, e.g. a unique determinant, from a protein represented by SEQ ID NO: 14. A further aspect of the present invention features a monoclonal or polyclonal antibody preparation specifically reactive with an epitope of the CR8 immunogen. Another aspect of the present invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes a CR8 polypeptide, analogues and/or fragments thereof. In certain preferred embodiments, the subject CR8 nucleic acid includes a transcriptional regulatory sequence, e.g., at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the CR8 gene sequence, e.g., to render the CR8 gene sequence suitable for use as an expression vector. In yet a further preferred embodiment, the nucleic acid which encodes a CR8 polypeptide of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least about 12 to 1,000, more preferably to at least about 20 to 500, more preferably to at least about 40 to 300consecutive nucleotides of SEQ ED NO: 13. In yet a further preferred embodiment, the CR8 encoding nucleic acid hybridizes to a nucleic acid probe corresponding to a subsequence encoding at least about 4 to 300, more preferably at least about 10 to 200, and even more preferably at least about 20 to 100 and longer amino acid residues between residues 1-412 of SEQ ID NO: 14. In preferred embodiments the nucleic acid sequence includes at least about 1, 2, 3 or 5, and preferably at least about 10, 20, 50, or 100 to 1,000 nucleotides from the region of SEQ ED NO: 13 which encodes amino acid residues 1-412 of SEQ ED NO: 14. The encoded peptide includes at least about 1, 2, 3, 5, 10, 20, or 30 to 350 amino acid residues from amino acid residues 1-412 of SEQ ID NO: 14.
DNA and peptide analogues and fragments of analogues and original DNA and polypeptide CR8 sequences are also part of the invention, in the form described above for CRI. There is significant sequence homology (about 40 to 45%) within an N-terminal 58 amino acid residue region to transcription factors that have a basic-Helix-Loop-Helix (bHLH) motif. The protein encoded by the bHLH region of the gene has been expressed in E. coli and has been found to bind to a hexanucleotide prediαed by the binding specificity of other bHLH proteins. (See Feder, J. et al., Moi. Cell Biol. 13(1): 105-113 (1993)). The N-terminal basic region binds to DNA and the HLH region serves as a protein dimerization motif. From the sequence of the bHLH region, CR8 fits into a class by itself. It shares most homology with Drosophila transcription repressors of the hairy family. However, CR8 lacks amino acid residues in the basic region and a C-terminal WRPW motif, characteristic for hairy proteins. CR8 also binds to Class B E-box sites (CACGTC/CATGTG), as do the c-myc family of bHLH proteins, rather than to Class C sites (CAGCCG) preferred by hairy-related family members. CR8 is ubiquitously expressed in all tissues examined except placenta. Its expression is induced by cytokines such as IL-2 and IL-3, which stimulate cellular proliferation, and also by IFNβ and elevated cAMP, which antagonize proliferation. Because CR8 contains a bHLH domain, it is most likely a protein that binds to DNA and modifies gene expression, either by aαivation or by suppression. Since CR8 binds to class B E-bax sequences, which the proto-oncogene c-myc family members also bind, it is likely that CR8 modifies the expression of genes important for the intermediate and late phases of ligand-promoted cell cycle progression. It follows that CR8 is a prime candidate for the development of new assays to discover agents that modify cellular function by either enhancing or suppressing CR8 gene expression or CR8 funαion. The CR8 gene and its gene produα are described in further detail below in Example VII. In summary, of the eight CR genes isolated using the thiol-seleαed IL-2-induced cDNA library, two are DNA binding proteins, one is a newly recognized transmembrane receptor, one contains an SH2 domain, one has homology to a newly recognized family of small proteins that regulate cellular proliferation, and another is a serine/threonine kinase known to be IL-2-induced, and to be over-expressed in MuLV-induced T cell lymphomas. Allowing for redundancies, a conservative estimate is that there are still about 40 to 50 novel genes induced by IL-2, which can be isolated using the method of the present invention.
Accordingly, the present invention pertains to an isolated nucleic acid comprising all redundant nucleotide sequences encoding the CR proteins, homologues, analogues and muteins described below. In one preferred embodiment, the invention comprises nucleotide sequence encoding anyone of the CR proteins, e.g., CRI, CR2, CR3, CR4, CR5, CR6, CR7, and CR8, particularly those corresponding to CRI, CR2, CR3, CR5, CR6, and CR8 and/or equivalents, homologues, analogues, and fragments of such nucleic acids and of CR4 and CR7.
The term "nucleic acid" as used herein is intended to include fragments and equivalents. The term "equivalent" as used herein refers to nucleotide sequences encoding funαionally equivalent CR proteins or functionally equivalent peptides which retain other activities of an CR protein such as described herein. Equivalent nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants and include, therefore, sequences that differ from the nucleotide. sequence CR proteins shown in any of SEQ. ID Nos: 2, 4, 6, 8, 10, 12, 14, and 28, due to the degeneracy of the genetic code. Equivalents also include nucleotide sequences that hybridize under stringent conditions (i.e., equivalent to about 20-27°C below the melting temperature (Tm) of the DNA duplex formed in about IM salt) to the nucleotide sequence encoding the presently claimed CR proteins represented as SEQ. ID Nos: 2, 4, 6, 8, 10, 12, 14, and 28. In one embodiment, equivalents further include nucleic acid sequences derived from and evolutionarily related to, any of the coding fragments of nucleotide sequences shown as SEQ. ID Nos: 1, 3, 5, 7, 9, 11, 13, and 27. Moreover, it is explicitly contemplated by the present invention that, under certain circumstances, it may be advantageous to provide homologs of the subjeα CR proteins which have at least one biological aαivity of a CR protein. Such homologs of the subjeα CR proteins can be generated by mutagenesis, such as by discrete point mutation(s) or by truncation. For instance, mutation can give rise to homologs which retain substantially the same, or merely a subset, of the biological aαivity of the CR protein from which it was derived. Alternatively, antagonistic forms of the protein can be generated which are able to inhibit the funαion of the naturally occurring form of the CR protein. A protein has CR biological activity if it has one or more of the following properties: (1) its expression is regulated by ligand-receptor stimulation; and (2) it participates in ligand-receptor modification of cellular function, e.g. proliferation, differentiation, programmed cell death.
As used herein, the term "gene" or "recombinant gene" refers to a nucleic acid comprising an open reading frame encoding a CR protein of the present invention, including both exon and (optionally) intron sequences. A "recombinant gene" refers to nucleic acid encoding a CR protein and comprising CR encoding exon sequences, though it may optionally include intron sequences which are either derived from a chromosomal CR gene or from an unrelated chromosomal gene. The term "intron" refers to a DNA sequence present in a given CR gene which is not translated into protein and is generally found between exons. As used herein, the term "transfeαion" means the introduαion of a nucleic acid, e.g., an expression veαor, into a recipient cell by nucleic acid-mediated gene transfer. "Transformation", as used herein, refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of the CR protein of the present invention or where anti-sense expression occurs from the transferred gene, the expression of a naturally-occurring form of the CR protein is disrupted. As used herein, the term "veαor" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of preferred veαor is an episome, i.e., a nucleic acid capable of extra- chromosomal replication. Preferred veαors are those capable of autonomous replication and/expression of nucleic acids to which they are linked. Veαors capable of direαing the expression of genes to which they are operatively linked are referred to herein as "expression veαors". In general, expression veαors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer to circular double stranded DNA loops which, in their veαor form are not bound to the chromosome. In the present application, "plasmid" and "veαor" are used interchangeably as the plasmid is the most commonly used form of veαor. However, the invention is intended to include such other forms of expression veαors which serve equivalent funαions and which become known in the an subsequently hereto. "Transcriptional regulatory sequence" is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked. In preferred embodiments, transcription of a recombinant CR gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell-type in which expression is intended. The recombinant gene can also be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring form of the CR proteins. As used herein, the term "tissue-specific promoter" means a DNA sequence that serves as a promoter, i.e., regulates expression of a seleαed DNA sequence operably linked to the promoter, and which effects expression of the seleαed DNA sequence in specific cells of a tissue. The term also covers so-called "leaky" promoters, which regulate expression of a seleαed DNA primarily in one tissue, but cause expression in other tissues as well.
As used herein, a "transgenic animal" is any animal, preferably a non-human mammal, e.g. a rat, a mouse or pig, in which one or more of the cells of the animal includes a transgene. The transgene is introduced into the cell, directly or indirectly by introduαion into a precursor of the cell, by way of deliberate genetic manipulation, such as by micro injeαion or by infeαion with a recombinant virus. The language "genetic manipulation" does not include classical cross¬ breeding, or in vitro fertilization, but rather is direαed to the introduαion of a recombinant DNA molecule. This molecule can be integrated within a chromosome, or it may be extrachromosomally replicating DNA. In the transgenic animals described herein, the transgene causes cells to express a recombinant form of one or more of the subject CR proteins, or alternatively, .to disrupt expression of one or more of the naturally-occurring forms of the CR genes. As used herein, the term "transgene" refers to a nucleic acid sequence which is partly or entirely heterologous, i.e., foreign, to the animal or cell into which it is introduced, or, is homologous to an endogenous gene of the animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid. As is well known, genes for a particular polypeptide may exist in single or multiple copies within the genome of an individual. Such duplicate genes may be identical or may have certain modifications, including nucleotide substitutions, additions or deletions, which all still code for polypeptides having substantially the same aαivity. The term "DNA sequence encoding a CR protein" refers to one or more genes within a particular individual. Moreover, certain differences in nucleotide sequences may exist between individual organisms, which are called alleles. Such allelic differences may or may not result in differences in amino acid sequence of the encoded polypeptide yet still encode a protein with the same biological aαivity. "Cells," "host cells" or "recombinant host cells" are terms used interchangeably herein. Such terms refer not only to the particular subjeα cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A "chimeric protein" or "fusion protein" is a fusion of a first amino acid sequence encoding one of the subject CR proteins with a second amino acid sequence defining a domain foreign to and not substantially homologous with any domain of the subject CR protein. A- chimeric protein may present a foreign domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it may be an "interspecies", "intergeneric", etc. fusion of protein struαures expressed by different kinds of organisms. The language "evolutionarily related to", with respect to nucleic acid sequences encoding CR proteins, refers to nucleic acid sequences which have arisen naturally in an organism, including naturally occurring mutants. This language also refers to nucleic acid sequences which, while derived from a naturally occurring CR nucleic, have been altered by mutagenesis, as for example, combinatorial mutagenesis, yet still encode polypeptides which have at least one aαivity of a CR protein.
Thus, the present invention provides a substantially pure polynucleotide comprising a nucleic acid encoding a polypeptide having one or more of the charaαeristics and/or runαionalities of anyone of the CR proteins, including CRI, CR2, CR3, CR4, CR5, CR6, CR7 and CR8 proteins, analogues, homologues, fragments, and combinationes thereof. In one embodiment, the nucleic acid comprises a cDNA encoding a peptide having at least one aαivity of a subjeα CR proteins. Preferably, the nucleic acid comprises a DNA molecule comprising at least a portion of anyone of the nucleotides SEQ. ID Nos: 1, 3, 5, 7, 9, 11, 13, and 27. Preferred portions of these cDNA molecules include the coding regions of the genes, fragments thereof, and combinations thereof. Preferred nucleic acids encode a CR protein comprising an amino acid sequence about 60% to 99% homologous, preferably at least about 60% homologous, more preferably about 70% homologous and most preferably about 80%, 90%, or 95% homologous to one of anyone of the amino acid sequences SEQ. ID Nos: 2, 4, 6, 8, 10, 12, 14, or 28, or combinations or fragments thereof. Nucleic acids which encode polypeptides having an activity of a subjeα CR protein and having at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homology to anyone SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, or 28, are also within the scope of the invention. The term "homology" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a funαion of the number of matching or homologous positions shared by the sequences. Another aspeα of the invention provides a nucleic acid which hybridizes under high or low stringency conditions to a nucleic acid which encodes a peptide having all or a portion of amino acid SEQ. ID NO: 2, SEQ. ID NO: 4, SEQ. ID NO: 6, SEQ. ID NO: 8, SEQ. ID NO:
10, SEQ. ID NO: 12, SEQ. ID NO: 14, or SEQ. ID No: 28. Appropriate stringency conditions which promote DNA hybridization, for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y., 6.3.1-6.3.6 (1989). For example, the salt concentration in the wash step can be seleαed from a low stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2 x SSC at 50°C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C.
Nucleic acids having a sequence that differs from the nucleotide SEQ ID Nos: 1, 3, 5, 7, 9,
11, 13, and 27, due to degeneracy or redundancy of the genetic code, are also within the scope of the invention. Such nucleic acids encode functionally equivalent peptides , i.e., a peptide having a biological activity of a CR protein, but differ in sequence from the sequence shown in said sequence listings due to degeneracy in the genetic code. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC each encode histidine) may result in "silent" mutations which do not affect the amino acid sequence of the CR protein. However, it is expeαed that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject CR proteins will exist among vertebrates. One skilled in the art will appreciate that these variations in one or more nucleotides (up to about 3 to 5% of the nucleotides) of the nucleic acids encoding polypeptides having an activity of an CR protein may exist among individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention. Fragments of the nucleic acids encoding the aαive portion of the presently claimed CR proteins are also within the scope of the invention. As used herein, a fragment of the nucleic acid encoding the aαive portion of a CR protein refers to a nucleic acid having fewer nucleotides than the nucleotide sequence encoding the entire amino acid sequence of a CR protein but which nevertheless encodes a peptide having a biological aαivity of the CR proteins described herein. Nucleic acid fragments within the scope of the present invention include those capable of hybridizing under high or low stringency conditions with nucleic acids from other species for use in screening protocols to deteα CR homologs, as well as those capable of hybridizing with nucleic acids from human specimens for use in deteαing the presence of a nucleic acid encoding one of the subjeα CR proteins, including alternate isoforms, e.g. mRNA splicing variants. Nucleic acids within the scope of the invention can also contain linker sequences, modified restriction endonuclease sites and other sequences useful for molecular cloning, expression or purification of recombinant forms of the subject CR proteins. A nucleic acid encoding a peptide having an aαivity of an CR protein can be obtained from mRNA present in any of a number of eukaryotic cells. Nucleic acids encoding CR proteins of the present invention can also be obtained from genomic DNA obtained from both adults and embryos. For example, a gene encoding a CR protein can be cloned from either a cDNA or a genomic library in accordance with protocols herein described, as well as those generally known to persons skilled in the art. A cDNA encoding one of d e subject CR proteins can be obtained by isolating total mRNA from a cell, e.g. a mammalian cell, e.g. a human cell, including tumor cells. Double stranded cDNAs can then be prepared from the total mRNA, and subsequently inserted into a suitable plasmid or baαeriophage veαor using any one of a number of known techniques. The gene encoding the CR protein can also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention. The nucleic acid of the invention may be a DNA or an RNA. Preferred nucleic acids are the DNAs represented by the sequences shown in SEQ. ID Nos: 1, 3, 5, 9, 11, 13, and homologues thereof and of SEQ. ID Nos: 7 and 27, and coding fragments thereof.
This invention also provides expression vectors containing a nucleic acid encoding a peptide having an aαivity of an CR protein, operably linked to at least one transcriptional regulatory sequence. The language "operably linked" refers to linkage of the nucleotide sequence to a regulatory sequence in a manner which allows expression of the nucleotide sequence. Regulatory sequences are art-recognized and are seleαed to direct expression of the peptide having an aαivity of a CR protein. Accordingly, the language "transcriptional regulatory sequence" includes promoters, enhancers and other expression control elements. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). The design of the expression veαor may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. In one embodiment, the expression vector includes a recombinant gene encoding a peptide having an aαivity of a subjeα CR protein. Such expression veαors can be used to transfeα cells and thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein. Moreover, such veαors can be used as a part of a gene therapy protocol to reconstitute the function of, or alternatively, abrogate the funαion of one of the subject CR proteins in a cell in which a CR protein is misexpressed.
Another aspeα of the present invention concerns recombinant forms of the subject CR proteins which are encoded by genes derived from eukaryotic organisms, e.g. mammals, e.g. humans, and which have at least one biological activity of a CR protein. The term "recombinant protein" refers to a protein of the present invention which is produced by recombinant DNA techniques, wherein generally DNA encoding the subjeα CR protein is inserted into a suitable expression veαor which is in turn used to transform a host cell to produce the heterologous protein. Moreover, the phrase "derived from", with respeα to a recombinant gene encoding the recombinant CR protein, includes within the meaning of "recombinant protein" those proteins having an amino acid sequence of a native CR protein of the present invention, or an amino acid sequence similar thereto which is generated by mutations including substitutions and deletions (including truncation) of a naturally occurring CR protein of a organism. Preferred recombinant proteins in accordance with the present invention, in addition to native CR proteins, are those about 60 to 99% homologous, preferably at least about 60% homologous, more preferably at least about 70% homologous and most preferably at least about 80% homologous to any one of amino acid SEQ. ID Nos: 2, 4,. 6, 8, 10, 12, 14, or 28. Polypeptides having an activity of the subjeα CR proteins and having about 60% to 99% homology, preferably at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homology to anyone of SEQ. ID Nos.: 2, 4, 6, 8, 10, 12, 14, or 28, are also within the scope of the invention. The present invention further pertains to recombinant forms of the subjeα CR proteins which are encoded by genes derived from an organism and which have amino acid sequences evolutionarily related to a CR protein of either SEQ. ID NO: 2, 4, 6, 8, 10, 12, or 14. The language "evolutionarily related to," with respeα to amino acid sequences of the present recombinant CR proteins, refers to CR proteins having amino acid sequences which have arisen naturally, as well as mutational variants of CR proteins which are derived, for example, by combinatorial mutagenesis. Preferred evolutionarily derived CR proteins are at least 60% homologous, more preferably at least about 70% homologous and most preferably at least about 80% homologous to anyone of SEQ. ID NO: 2, SEQ. ID NO: 4, SEQ. ID NO: 6, SEQ. ID NO: 8, SEQ. ID NO: 10, SEQ. ID NO: 12, SEQ. ID NO: 14, or SEQ. ID NO: 28. Polypeptides having about 60 to 99% homology, preferably at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homology with a sequence shown in any of SEQ ID Nos: 2, 4, 6, 8, 10, 12, or 14 are also within the scope of the invention.
The present invention further pertains to methods of producing the subjeα CR proteins. For example, a host cell transfeαed with a nucleic acid vector direαing expression of a nucleotide sequence encoding the subject CR protein can be cultured under appropriate conditions to allow expression of the peptide to occur. The peptide can be secreted and isolated from a mixture of cells and medium containing the recombinant CR peptide. Alternatively, the peptide can be retained cytoplasmically and the cells harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproduαs. Suitable media for cell culture are well known in the art. The recombinant CR peptide may be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, eleαrophoresis, and immunoaffinity purification with antibodies specific for such peptide. In a preferred embodiment, the recombinant CR protein is a fusion protein containing a domain which facilitates its purification.
This invention also pertains to a host cell transfeαed to express a recombinant form of at least one of the subjeα CR proteins. The host cell can be any prokaryotic or eukaryotic cell. Thus, a nucleotide sequence derived from the cloning of the CR proteins of the present invention, encoding all or a seleαed portion of a protein, can be used to produce a recombinant form of a CR protein via microbial or eukaryotic cellular processes. Ligating the polynucleotide sequence into a gene construα, such as an expression veαor, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, inseα or mammalian) or prokaryotic (baαerial cells), are standard procedures used in producing other well-known proteins, e.g. insulin, interferons, human growth hormone, IL-1, IL-2, and the like. Similar procedures, or modifications thereof, can be employed to. prepare recombinant CR proteins, or portions thereof, by microbial means or tissue-culture technology in accordance with the subjeα invention.
A recombinant CR gene may be produced by ligating a nucleic acid encoding anyone of thet CR proteins, or fragments thereof, into a veαor suitable for expression in either prokaryotic cells, eukaryotic cells, or both. Expression veαors for produαion of recombinant forms of the subjeα CR proteins include plasmids and other veαors. For instance, suitable veαors for the expression of a CR protein include plasmids of the types: pBR322-derived plasmids, pEMBL- derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli. A number of vectors exist for the expression of recombinant proteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduαion of genetic constructs into S. cerevisiae (see, for example, Broach et al., in Experimental Manipulation of Gene Expression, Ed., M. Inouye Academic Press, p. 83 (1983), incorporated by reference herein). These veαors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid. In addition, drug resistance markers such as ampicillin can be used. Preferred mammalian expression veαors contain both prokaryotic sequences to facilitate the propagation of the vector in baαeria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived veαors are examples of mammalian expression veαors suitable for transfection of eukaryotic cells. Some of these veαors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance seleαion in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: Chapters 16 and 17 (1989)). In some instances, the recombinant CR protein can be expressed using a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived veαors (such as the β-gal containing pBlueBac III).
When expression of a portion of one of the subjeα CR proteins is desired, i.e. a truncation mutant, it may be necessary to add a start codon (ATG) to the oligonucleotide fragment containing the desired sequence to be expressed. It is well known in the art that a methionine at the N-terminal position can be enzymatically cleaved by the use of the enzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli (Ben-Bassat et al., J. Baαeriol. 169: 751-757 (1987)) and Salmonella typhimurium and its in vitro aαivity has been demonstrated on recombinant proteins (Miller et al., P.N.A.S. (USA) 84: 2718-1722 (1987)). Therefore, removal of an N-terminal methionine, if desired, can be achieved either in vivo by expressing CR-derived polypeptides in a host which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or in vitro by use of purified MAP (e.g., procedure of Miller et al., supra). Alternatively, the coding sequences for the polypeptide can be incorporated as a part of a fusion gene including a nucleotide sequence encoding a different polypeptide. This type of expression system can be useful under conditions where it is desirable to produce an immunogenic fragment of a CR protein. The nucleic acid sequences corresponding to the portion of a subject CR protein to which antibodies are to be raised can be incorporated into a fusion gene construα which includes coding sequences for a late vaccinia virus structural protein to produce a set of recombinant viruses expressing fusion proteins comprising a portion of the CR protein as part of the virion. It has been demonstrated with the use of immunogenic fusion proteins utilizing the Hepatitis B surface antigen fusion proteins that recombinant Hepatitis B virions can be utilized in this role as well. Similarly, chimeric constructs coding for fusion proteins containing a portion of a CR protein and the poliovirus capsid protein can be created to enhance immunogenicity of the set of polypeptide antigens (see, for example, EP Publication No: 0259149; and Evans et al.. Nature 339: 385 (1989); Huang et al., J. Virol. 62: 3855 (1988): and Schlienger et al., J. Virol. 66: 2 (1992)). The Multiple Antigen Peptide system for peptide-based immunization may also be utilized to generate an immunogen, wherein a desired portion of a subjeα CR protein is obtained direαly from organo-chemical synthesis of the peptide onto an oligomeric branching lysine core (see, for example, Posnett et al., J.B.C. 263: 1719 (1988), and Nardelli et al., J. Immunol. 148: 914 (1992)). Antigenic determinants of me subjeα CR proteins can also be expressed and presented by bacterial cells.
In addition to enhancing immunogenicity, fusion proteins can also facilitate the expression of proteins, such as any one of the CR proteins of the present invention. For example, a CR protein of the present invention can be generated as a glutathione-S-transferase (GST- fusion protein). Such GST fusion proteins can enable easy purification of the CR protein, such as by the use of glutathione-derivatized matrices (see, for example, Current Protocols in Molecular Biology, eds. Ausabel et al., N. Y., John Wiley & Sons (1991)). Techniques for making fusion genes are known to those skilled in the art. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriαion enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, Eds. Ausubel et al., John Wiley & Sons (1992)).
Another aspeα of the invention pertains to isolated peptides having an aαivity of one of the subjeα CR proteins. In preferred embodiments, a biological aαivity of a CR protein includes: promotion of cell cycle progression (e.g., CRI); ligand-receptor signaling (e.g., CR3); cellular maturation, differentiation, and proliferation (e.g., CR4); enhancement or suppression of DNA replication (e.g., CR5, CR6); promotion of mRNA transcription by stimulating elongation of mRNA transcription (e.g., CR5, CR6); and transcriptional aαivation and repression (e.g., CR8). Other biological aαivities of the subjeα CR proteins are described herein or will be reasonably apparent to those skilled in the art. A polypeptide having at least one biological aαivity of the subject CR proteins may differ in amino acid sequence from the sequence shown in either SEQ. ID NO: 2, 4, 6, 8, 10, 12, or 14, but such differences result in a modified polypeptide which funαions in the same or similar manner as the native CR or which has the same or similar charaαeristics of the native CR protein. Various modifications of a CR protein of the present invention to produce these and other funαionally equivalent peptides are described in detail herein. The terms peptide, protein, and polypeptide are used interchangeably herein. The present invention also pertains to isolated CR proteins which are isolated from, or otherwise substantially free of other cellular proteins normally associated with the CR protein. The language "substantially free of other cellular proteins" (also referred to herein as "contaminating proteins") or "substantially pure, substantially pure preparation, or purified preparations" are defined as encompassing CR protein preparations having less than about 20% (by dry weight) contaminating protein, and preferably having less than about 5% contaminating protein. Funαional forms of the subjeα CR proteins can be prepared, for the first time, as purified preparations by using a cloned gene as described herein. As used herein, the term "purified", when referring to a peptide or DNA or RNA sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules, such as other proteins. The term "purified" as used herein preferably means at least about 80% by dry weight, more preferably in the range of about 95 to 99% by weight, and most preferably at least about 99.8% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present). The term "pure" as used herein preferably has the same numerical limits as the term "purified". "Isolated" and "purified" do not encompass either natural materials in their native state or natural materials that have been separated into components (e.g., in an acrylamide gel) but not obtained either as pure (e.g. lacking contaminating proteins, or chromatography reagents such as denaturing agents and polymers, e.g. acrylamide or agarose) substances or solutions. The term "isolated" as also used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, respeαively, that are present in the natural source of the macromolecule. For example, an isolated nucleic acid encoding one of the subject CR proteins preferably includes no more than 10 Kilobases (Kb) of nucleic acid sequence which naturally immediately flanks that particular CR gene in genomic DNA, more preferably no more than 5 Kb of such namrally occurring flanking sequences, and most preferably less than 1.5 Kb of such naturally occurring flanking sequence. The term isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an "isolated nucleic acid" is meant to include nucleic acid fragments which are not namrally occurring as fragments and would not be found in the natural state. Furthermore, isolated peptide portions of the subject CR proteins can also be obtained by screening peptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such peptides. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, a CR protein of the present invention can be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments which can funαion as either agonists or antagonists of a CR protein aαivity, such as by microinjeαion assays.
The structure of the subject CR proteins may be modified for such purposes as enhancing therapeutic or prophylaαic efficacy, or stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo). Such modified peptides, when designed to retain at least one activity of the naturally-occurring form of the protein, are considered funαional equivalents of the CR proteins described in more detail herein. Such modified peptide can be produced, for instance, by amino acid substitution, deletion, or addition. Moreover, it is reasonable to expeα that an isolated replacement in CR proteins of the invention of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (i.e. conservative mutations) will not have a major effect on the biological aαivity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are divided into four families: (1) acidic = aspartate, glutamate; (2) basic = lysine, arginine, histidine; (3) non-polar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In similar fashion, the amino acid repertoire can be grouped as (1) acidic = aspartate, glutamate; (2) basic = lysine, arginine histidine, (3) aliphatic = glycine, alanine, valine, leucine, isoleucine, serine, tlireonine, with serine and threonine optionally be grouped separately as aliphatic-hydroxyl; (4) aromatic = phenylalanine, tyrosine, tryptophan; (5) amide = asparagine, glutamine; and (6) sulfur - containing = cysteine and methionine. (see, for example, Biochemistry, 2nd ed., Ed., L. Stryer, WH Freeman and Co. (1981)). Whether a change in the amino acid sequence of a peptide results in a functional CR homolog can be readily determined by assessing the ability of the variant peptide to produce a response in cells in a fashion similar to the wild-type CR protein or peptide. Peptides in which more than one replacement has taken place can readily be tested in the same manner.
Another aspect of the invention pertains to an antibody or antibody preparation specifically reaαive with at least one epitope of at least one of the subject CR proteins. For example, by using immunogens derived from the present CR proteins, based on the cDNA sequences, anti-protein anti-peptide antisera or monoclonal antibodies can be made by standard protocols (see, for example, Antibodies: A Laboratory Manual, Ed., Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide, e.g., CR protein or an antigenic fragment which is capable of eliciting an antibody response. Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or odier techniques well known in the art. An immunogenic portion of the subject CR proteins can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies. In a preferred embodiment, the subject antibodies are immunospecific for antigenic determinants of the CR proteins of the present invention, e.g. antigenic determinants of a protein represented by one of SEQ. ID Nos: 2, 4, 6, 8, 10, 12, 14, or 28, or a closely related human or non-human mammalian homologue, e.g., at least about 90% homologous, more preferably at least about 95% homologous. In yet a further preferred embodiment, the anti-CR protein antibodies do not substantially cross react, i.e., reaα specifically, with a protein which is, e.g., less than about 90% homologous to one of SEQ. ID Nos: 2, 4, 6, 8, 10, 12, 14, or 28, e.g., less than about 95% homologous to one of SEQ. ID Nos: 2, 4, 6, 8, 10, 12, 14, or 28, e.g., less than about 98 to 99% homologous to one of SEQ. ID Nos: 2, 4, 6, 8, 10, 12, 14, or 28. The language "not substantially cross-reaα" means that the antibody has a binding affinity for a non-homologous protein which is less than about 10%, more preferably less than about 5%, and even more preferably less than about 1 %, of the binding affinity for a protein of SEQ. ID Nos: 2, 4, 6, 8, 10, 12, 14, or 28. Following immunization, anti-CR antisera may be obtained and, if desired, polyclonal anti-CR antibodies isolated from the serum. To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Such techniques are well known in the art, an include, for example, the hybridoma technique (originally developed by Kohler and Milstein, (1975) Nature, 256: 495-497), the human B cell hybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be screened immunochemically for produαion of antibodies specifically reaαive with a CR protein of the present invention and monoclonal antibodies isolated from a culture comprising such hybridoma cells. The term "antibody" as used herein is intended to include fragments thereof which are also specifically reaαive with one of the subject CR proteins. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Antibodies of the present invention are further intended to include bi-specific and chimeric molecules having an anti-CR portion. Both monoclonal and polyclonal antibodies (Ab) direαed against CR or CR variants, and antibody fragments such as Fab' and F(ab')2, can be used to block the action of a CR proteins and allow the study of the role of the particular CR protein of the present invention in cell signaling.
The nucleotide sequence determined from the cloned cDNA of the CR proteins from a human cell line permits the preparation of probes for identification of CR homologues in other human cell types, as well as CR homologues from other animals. For instance, the present invention also provides a probe/primer comprising a substantially purified oligonucleotide, wherein the oligonucleotide comprises a region of nucleotide sequence which hybridizes under stringent conditions to at least about 10 consecutive nucleotides of sense or anti-sensenucleic acids of SEQ. ID Nos: 1, 3, 5, 7, 9, 11, 13, or 27, or naturally occurring mutants thereof. In preferred embodiments, the probe/primer further comprises a label group attached thereto, which may be deteαed, e.g., the label group may be seleαed from radioisotopes, fluorescent compounds, enzymes, and enzyme co-faαors. Such probes can be used as a part of a test kit for measuring a level of an CR nucleic acid in a sample of cells from a patient, e.g., measuring a CR mRNA level; e.g. determining whether a genomic CR gene has been mutated or deleted. In addition, nucleotide probes can be generated from the cloned sequence of the subjeα CR proteins, which allow for histological screening of intaα tissue and tissue samples for the presence of a CR mRNA. Use of probes direαed to CR mRNAs, or to genomic CR sequences, can be used for both prediαive and therapeutic evaluation of allelic mutations which might be manifest in a variety of disorders including cancer, immunodeficiencies, autoimmune disorders, developmental abnormalities, infeαious diseases, toxic damage due to irradiation, chemicals, and other noxious compounds or natural products. Used in conjunαion with anti-CR antibody immunoassays, the nucleotide probes can help facilitate the determination of the molecular basis for a developmental disorder which may involve some abnormality associated with expression (or lack thereof) of a CR protein. For instance, variation in CR synthesis can be differentiated from a mutation in the CR coding sequence.
Also, the use of anti-sense techniques, e.g., microinjeαion of antisense molecules, or transfeαion with plasmids whose transcripts are anti-sense with regard to a CR mRNA or gene sequence, may be used to investigate the normal cellular funαion of each of the novel CR proteins, e.g. in cell signaling or as therapeutic agents to tie-up undesirable gene transcription. Such techniques can be utilized in cell culture, but can also be used in the creation of transgenic animals. Furthermore, by making available purified and recombinant CR proteins, the present invention facilitates the development of assays which can be used to screen for drugs which are either agonists or antagonists of the normal cellular function of the subjeα CR proteins, or of their role in cell signaling.
In another aspeα, the invention feamres transgenic non-human animals which express a recombinant CR gene of the present invention, or which have had one or more of the subject CR gene(s), e.g., heterozygous or homozygous, disrupted in at least one of the tissue or cell-types of the animal. In yet another aspect, the invention features an animal model for disorders related to cell signaling, which has a CR allele which is mis-expressed. For example, a mouse bred to have a CR allele deleted, or in. which all or part of one or more CR exons are deleted or mutated. Such a mouse model is suitable for smdying disorders arising from mis-expressed CR genes.
This invention is further illustrated by the following examples, which in no way should be construed as being further limiting. The contents of all cited references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited tiiroughout this application are hereby expressly incorporated by reference.
EXAMPLES
Example I: Construction of cDNA Library Containing
Clones of Ligand-induced Genes Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll/Hypaque discontinuous centrifugation, and cultured at 10^ cells/ml in complete medium comprised of RPMI 1640 (GIBCO Laboratories, Grand Island, NY) supplemented with 10% heat-inactivated (56°C, 30 min) calf serum (Sterile Systems, Inc., Logan, UT), 50 mg/ml L-glutamine, and 50 units/ml penicillin. T-cells were activated by stimulation of the CD3 component of the T-cell receptor complex with an anti-CD3 reaαive monoclonal antibody (OKT3, 1:10,000 dilution, Ortho Pharmaceuticals, Raritan, NJ) in the presence of absence of 10 mg/ml CHX, and DNA synthesis was monitored at 48-52 hr by adding 0.5 mCi [^H -thymidine to 200 ml aliquots of cell cultures in 96-well microtiter plates. Cultures were harvested onto glass fiber filters, radioaαivity was counted by liquid scintillation, and [^H]-_hymidine incorporation was calculated as cpm/104 cells/hr.
IL-2R-positive T-cell blasts were prepared by stimulation of PBMCs with OKT3 for 3 days, after which the cells were washed and replaced in culture for an additional 1 1 days in the presence of 500 pM IL-2. The cells were subsequently washed and placed in culture in the absence of IL-2 for 36 hr, followed by a 12 hr stimulation with 50 ng/ml phorbol 12,13 dibutyrate (PdBu) to augment high-affinity IL-2R expression. Cells were washed free of PdBU and placed in culture for 12 hr prior to restimulation. Such treatment enabled the generation of a GQ/GI -synchronized cell population, made up of greater than 90% T8-positive T lymphocytes (Gullberg et al., J. Exp. Med. 163: 270-284 (1986)).
Human IL-2R-positive T-cell blasts were cultured in the presence of 1 nM IL-2, 10 mg/ml CHX, 250 mM 4-thiouridine (Sigma Chemical Co., St. Louis, MO) and 2.5 mCi/ml [5,6- ]-uridine (48 Ci/mmole, Amersham, Arlington Heights, IL) for 2 hr. CHX was included in the 2 hr IL-2 stimulation of the IL-2R-positive, GQ/GI -synchronized human T-cells from which the cDNA library was generated in order to isolate immediate-early genes, and also to possibly superinduce the expression of low-abundance messages. Total RNA was isolated essentially as described by Caligiuri et al. (Caliguri et al., J. Exp. Med. 171: 1509-1526 (1989)), and the 4- thiouridine-labeled RNA purified by passage over a phenylmercury agarose column as described by Woodford et al. (Woodford et al, Anal. Biochem. 171: 166-172 (1988)). The cells were labeled with 4-thiouridine during stimulation, to enable isolation of only those transcripts which were synthesized during the period of IL-2 and CHX treatment (Stetler et al. (1984) Proc. Nat. Acad. Sci. (USA) 81: 1144-1148 and Woodford et al. (1988) Anal. Biochem. 171: 166-172). Fraαionation of total cellular RNA resulted in a 10-fold enrichment for newly-synthesized transcripts. This thiol-seleαed RNA was used in the synthesis of Not-1 primer/adapter-primed cDNA, utilizing the Riboclone cDNA Synthesis System (Promega, Madison, Wl) according to manufacturers instruαions. After addition of EcoRI adapters (Promega), Not-1 digestion, and size seleαion for fragments greater than 500 base pairs (bp), the cDNA was ligated direαionally into an EcoRI- and Not- 1 -digested p Bluescript II SK+ plasmid veαor (Stratagene, La Jolla, CA), followed by transformation into Epicurian Coli XL-1 Blue competent cells (Stratagene). A cDNA library of approximately 10,000 clones resulted.
Example II: Screening of cDNA Library for Clones
Containing Ligand-induced Genes
About 10% of the cDNA library was then screened using radiolabeled cDNA probes made from mRNA isolated from T-cells induced with IL-2 or from uninduced cells as follows. Single-stranded [32p]_iabeled cDNA probes were prepared from poly(A)+ RNA isolated from human T-cell blasts stimulated for 2 hr with medium (unstimulated probe), or 1 nM IL-2 and 10 mg/ml CHX (stimulated probe). Total cellular RNA was prepared as described by Caligiuri et al. (Caliguri et al., J. Exp. Med. 171 : 1509-1526 (1989)), and poly(A)+ RNA was isolated by three passages over an oligo-dT-cellulose column (5 Prime-3 Prime, West Chester, PA). First strand cDNA synthesis was performed with an oligo-dT 12-18 primer (United States Biochemical Corp., Cleveland, OH), using the Riboclone cDNA Synthesis System (Promega, Madison, Wl) according to manufacturers instruαions, with the exception of dCTP at a final concentration of 35 mM and the addition of 2.5 mCi/ml [3 P]-dCTP. Hybridization was carried out for 72-96 hr at 42°C in 50% formamide, with a final probe concentration of approximately 2 x 10^ cpm/ml (W. M. Strauss, in Current Protocols in Molecular Biology, pp. 6.3.1-6.3.6 (1989)). Subsequent to hybridization, filters were washed repeatedly at 62°C in 0.1 x SSC (1 x SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 0.1 % SDS and placed on film (Kodak XAR-5) with Dupont Cronex intensifying screens overnight at -70°C. The initial screening yielded 18 putative positive clones which exhibited differential hybridization to the stimulated and unstimulated probes after three independent screens. These clones were isolated for further characterization by Northern Blot analysis.
Total cellular RNA was isolated by the guanidine thiocyanate method described by Caligiuri et al. (ibid.), and denatured in glyoxal and DMSO. The RNA was fractionated on a 1 % agarose gel in 0.01 M NaH2PO4 with 0.5 mg/ml ethidium bromide (Selden, Current Protocols in Molecular Biology, pp. 4.9.5-4.9.8 (1989)). To estimate sizes of RNA transcripts, a 0.24-9.5 Kb RNA ladder (Bethesda Research Laboratories, Gaithersburg, MD) was run alongside the cellular RNA samples. After visualization under ultraviolet light, the RNA was transferred to nitrocellulose by capillary transfer in 10 x SSC. Plasmids were purified from the clones of interest, and the Not-1- and EcoRI -excised inserts [32p]_iabeled with random primers. Hybridization was carried out in 50% formamide at 42°C for 48-72 hr, followed by repeated washes in 0.1 X SSC, 0.1 % SDS at 56-62°C (Selden, ibid.). Filters were exposed to Kodak XAR-5 film with Dupont Cronex intensifying screens, and specific bands quantitated with an EC densitometer (EC Apparatus Corp., St. Petersburg, FL).
In as much as CHX was included in bom the library and probe preparation, it was essential to verify that the differential expression of putative clones observed upon colony screening was not due solely to the effeαs of this drug. In addition, determination of the sizes and patterns of induction of the RNA transcripts was necessary to enable estimation of the redundancy of the clones. Therefore, Northern blot analysis was performed with RNA isolated from human IL-2R-positive T-cell blasts stimulated with either CHX or IL-2 alone, or with a combination of the two agents.
Hybridization of the RNA with probes generated from the inserts of each of the 18 putative clones resulted in the identification of 4 clones that were solely CHX-induced. For the remaining 14 clones, the induαion by the combination of IL-2 and CHX could not be accounted for by the effeαs of CHX alone. Based upon the patterns of induαion and approximate sizes of the RNA transcripts, 8 readily distinguishable and apparently unique IL-2-induced genes were discerned, as partial sequences, among these 14. These are described in Table X below.
Table X: Some Characteristics of the Eight Proteins Cloned
Figure imgf000062_0001
The original designations of the CR clones are included in parentheses in the left-hand column of Table X above. The original designations are used herein to refer to the partial sequences shown in the column second from the left in Table II. As shown in Table II and in Figures 8A-8H, three of the genes, CRI, CR3, and CR5, were induced by IL-2 alone, while five of the genes, CR2, CR4, CR6, CR7, and CR8, were induced by both CHX and IL-2. In several instances, the combination of IL-2 and CHX resulted in a marked synergistic induction.
Example III: Kinetic Analysis of IL-2-Induced Gene Expression
The temporal expression of the novel, IL-2-induced genes was determined by Northern blot analysis, using RNA isolated from human IL-2R-positive T-cell blasts after IL-2 stimulation in the presence or absence of CHX. Northern blots were prepared with 15 mg total RNA isolated from GQ/GJ -synchronized human T-cells stimulated for 0, 0.5, 1, 2, 4, or 8 hours with 1 nM IL- 2 or IL-2 + 10 mg/ml CHX. Filters were probed with the cDNA inserts of the IL-2-induced clones. Two of the genes, 1A8 and 10D6, exhibited rapid induαion, reaching peak levels within 1-4 hr of IL-2 stimulation and returning to basal levels after 8 hr, while the other six clones remained at elevated levels for at least 8 hr after IL-2 treatment. The magnitude of IL-2 induαion of steady state RNA levels of the clones ranged from an approximately 5-fold elevation of clone 11B2 to a greater than 50-fold stimulation of clone 10F9 during the interval examined. These results are also summarized in Table X above. Several of the clones were superinduced by CHX, witii an increase observed in both the magnitude and duration of the IL-2 response. The kinetics of induαion of previously characterized IL-2-responsive genes have been found to range from those such as c-fos, which are rapidly and transiently induced within minutes of IL-2 stimulation (Dautry et al., J. Biol. Chem. 263: 17615-17620 (1988)), to those which remain at elevated levels through Gi to S phase entry (Sabath et al., J. Biol. Chem. 265: 12671-12678 (1990)).
Example IV: Sequence Analysis of Clones Containing Ligand-induced Genes
To verify the redundancy of the clones as estimated from Northern analysis, as well as to determine the identities of the genes, the cDNA clones were subjeαed to sequence analysis.
Plasmids were isolated from the clones of interest essentially as described by Kraft et al., Biotechniques 6: 544-547 (1988)), and veαor primers were used to sequence the termini of the cDNA inserts, employing the Sequenase 2.0 dideoxy sequencing kit (United States Biochemical, Cleveland, OH). Approximately 200 bases of sequence were attained from each end of the inserts. These partial sequences are described in Table II. Searches of the GenBank and EMBL data bases were performed with the FASTA program as described by Pearson et al., P.N.A.S. (USA) 85: 2444-2448 (1988).
The combination of sequence and Northern analyses revealed mat the 14 putative IL-2- induced clones consisted of 8 unique genes, three of which, 1A8, 11B2, and 13E2, were isolated three times each. Searches of the GenBank and EMBL data bases with the partial sequences enabled the identification of one clone, 11E6, as pim-1, a previously characterized IL-2-induced gene (Dautry et al. (1988) J. Biol. Chem. 263: 17615-17620; and Kakut-Houri et al. (1987) Gene 54: 105-111 which encodes a 33 kD cytoplasmic kinase (Telerman et al. (1988) Moi. Cell. Biol. 8: 1498-1503). Thus, by utilizing the method of the invention seven unique IL-2 induced genes were cloned, representing novel human genes. These clones were identified after screening only approximately 800 library colonies, and thus, it is estimated that as many as 80 additional novel IL-2-induced genes remain to be detected in the 10,000-clone library.
To determine the complete sequences of these clones described in Table X above the original partial cDNAs were used as probes to screen a second cDNA library. It is standard procedure to use partial cDNA inserts identified by an initial screen of a cDNA library to make radiolabeled cDNA probes to screen a second library to obtain clones with the portions missing in the initial cDNA clones. This was done, briefly, as follows: a second cDNA library was prepared from mRNA obtained from human T cells stimulated for two hours with interleukin-2 in the presence of cycloheximide by cloning into the λgt-10 phage vector using standard methods. (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, pp. 2.82-2.122 (1989)). This second cDNA library was then screened using as probes each of the cDNA fragments obtained from the first, thiol-selected cDNA library. Candidate clones that corresponded to the correct size according to the mRNA were then subcloned and sequenced. The complete cDNA sequences (and the prediαed amino acid sequences) of seven out of eight of these clones are set forth in SEQ. ID Nos: 1-14 and Figures 1- 7. The complete cDNA sequence (and the prediαed amino acid sequence) of the eighth clone was determined to be identical to that of the IL-2 induced gene pim-1. The nucleotide sequence as well as the prediαed amino acid sequence of pim-1 are set forth at page 605 in Selten, G. et al., Cell 46: 603-611 (1986).
Example V: Determination of Sensitivity of IL-2-Induced Gene Expression
As a further means of characterizing the regulation of expression of these genes, the sensitivity of induαion to the known IL-2 funαional antagonist was investigated. Human IL-2R- positive T-cell blasts were stimulated with IL-2 in the absence or presence of 0.5 mM dibutyryl- cAMP, a concentration of the membrane-permeant cAMP analog sufficient to inhibit IL-2- mediated G\ progression without adversely affecting cellular viability. The effeα of an equivalent molar amount of sodium butyrate, which does not inhibit the IL-2 response, was also tested to control for the aαions of free butyric acid. Northern blots were prepared as follows: Human IL-2R-positive T cells were treated with 1 nM IL-2 alone or in combination with 0.5 mM dibutyryl cAMP or sodium butyrate (NaBt) for 1, 2, or 4 hours. Filters were prepared with 15 mg total RNA and hybridized with cDNA inserts or the IL-2 induced clones.
These analyses demonstrate that the IL-2 induction of one gene, 1A8 (Figure 10A) is markedly inhibited when the intracellular level of cAMP is raised by the addition of dibutyryl cAMP, whereas the expression of two others, 10D6 and 13E2, is augmented approximately 3- fold. By comparison, the expression of five of the genes was not affeαed by elevated cAMP. Thus, the sequences in clone 1A8 may be involved in T-cell proliferation. The faα that not all genes were sensitive to cAMP indicated that the observed results were not due to non-specific effects, and furthermore that the previously documented down-regulation of IL-2R binding capacity by cAMP (Johnson et al., J. Immunol. 145: 1144-1151 (1990)) could not account for the inhibition of gene expression. Example VI: Determination of Role of T-cell Receptor Activation in the
Stimulation of Expression of IL-2-Induced Genes
In order to determine if activation of the T-cell receptor mediates the stimulation of expression of cytokine IL-2-induced genes, the following study was performed. Northern blots were prepared from 20 mg total cellular RNA isolated from human peripheral blood mononuclear cells (PBMCs) stimulated with a monoclonal antibody (OKT3) specific to the CD3 component of the T-cell antigen receptor complex. Blots were probed with cDNA inserts of the IL-2-induced clones. Data was determined as me mean _+ SEM (n=6).
By isolation of RNA at early time intervals, it was possible to identify those genes which were induced by T-cell receptor triggering in the absence of IL-2 effects. Only one of the genes, 10D6, exhibited heightened levels of expression after 2 hr of T-cell receptor activation, while the seven others were apparently insensitive to this stimulus. Two of the clones, 1F5 and 11B2, were undetectable, even after seven days of autoradiographic exposure of the Northern blots. Two other genes, 11E6 and 13E2, were expressed at relatively high levels regardless of the stimulus; aαivation with anti-CD3 did not induce RNA expression beyond the level observed by culture in medium alone. Identical results were obtained after 1 and 4 hr of stimulation.
To determine whether the cells were actually aαivated via CD3, aliquots of the cells were left in culture for 52 hr in the presence of 10 mg/ml CHX, alone, OKT3 alone, or OKT3 + CHX, after which cell cycle progression was monitored by [^HJ-thymidine incorporation into DNA, which indicated that the cells were sufficiently stimulated by anti-CD3. Thus, the T-cell receptor-induced expression of only one of the genes was comparable to that seen with IL-2 stimulation, while the expression of the seven others was unique to the IL-2 signaling pathway. Thus, the methods described herein to identify IL-2-induced gene successfully selected and enriched for these genes that are highly specific for cytokine (IL-2) activation. Of the 8 IL-2 induced G progression genes reported here, only one appears to also be induced during the T cell receptor-mediated competence phase of the cell cycle. Thus, while several genes such as c-fos, c-myc and c-raf-1 are known to be induced during both the initial GQ- G] and subsequent G S phase transitions, the expression of a number of IL-2-stimulated genes is unique to the latter event. In addition, the immediate-early genes reported here appear to define a class distinα from the IL-2-induced genes isolated by Sabath et al. (Sabath et al., J. Biol. Chem. 265: 12671-12678 (1990)). These investigators utilized a differential screening procedure to isolate genes expressed much later, at the Gj/S phase boundary in a murine T helper clone, which was stimulated with IL-2 for 20 hr in the absence of protein synthesis inhibitors. In this case, the expression of only 3 of the 21 clones isolated was inhibited by CHX, while the remainder were insensitive to this agent. This pattern of regulation markedly contrasts with the CHX superinduαion observed with the immediate-early IL-2-induced genes described here. Moreover, these observations indicate that IL-2 stimulates a complex program of gene expression, ranging from those genes induced very early in Gj through those subsequently expressed at the Gi/S phase transition, which occur between about 18 and 36 hours after IL-2 stimulation.
Example VII: Cloning and Analysis of CR8 As described above, the CR8 gene encodes a novel basic helix-loop-helix (bHLH) protein.
While the CR8 transcript is ubiquitously expressed in many tissues, it is induced by IL-2 as well as by IL-3 in cytokine-dependent lymphoid cell lines. In an IL-2-dependent human T cell line Kit 225, the CR8 transcript is induced not only by IL-2, but also by interferon b and forskolin, which elevates intracellular cAMP. The bHLH domain of CR8 shows the highest structural homology to a Drosophila transcriptional repressor hairy. The recombinant CR8 protein binds preferentially to the Class B E-box DNA sequence (CACGTG), which is found in the promoter/enhancer regions of a number of genes associated with cell growth and differentiation, suggesting that CR8 may regulate the transcription of such genes.
The cloning of the full-length cDNA for CR8 is described in detail herein. The prediαed amino acid sequence revealed that CR8 contains a helix-loop-helix (HLH) domain, charaαeristic for transcription faαors. The HLH domain is a dimerization motif charaαerized by the two amphipathic α-helices separated by a non-helical loop of variable length (Davis, R.L. et al., Cell 60: 733-746 (1990)). Most of the HLH family members possess a cluster of basic amino acid residues immediately N-terminal to the HLH region [basic helix-loop-helix (bHLH)], which are required for site-specific DNA binding, while others lack the basic region and funαion as negative regulators of DNA binding (Benezra, R., et al., Cell 61: 49-59 (1990); Ellis, H.M. et al., Cell 61: 27-38 (1990); Garrell, J. et al., Cell 61: 39-48.22,28 (1990)). A wide variety of developmental processes are regulated by HLH proteins; the MyoD family of myogenic transcription factors direαly induce the expression of muscle-specific genes, thereby funαioning as master regulators of muscle cell lineage specification (reviewed in (Edmonson, D. G. et al. (1993) J. Biol. Chem. 268: 755-758; Weintraub, H., Cell 75: 1241-1244 (1993)). The crucial role of the bHLH protein encoded by the tal-1/SCL gene in hematopoiesis, originally discovered as a chromosomal breakpoint in leukemia (Begley, C. G. et al., P.N.A.S. (USA) 86: 10128-10132 (1989); Chen, G. et al., EMBO J. 9: 415-424 (1990); Finger, L. et al., P.N.A.S. (USA) 86: 5039-5043 (1989)), is illustrated by the absence of blood formation in tal-1 null mutant mice (Shivdasani, R.A. et al., Nature 373: 432-434 (1995)).
The regulation of immunoglobulin (Ig) gene expression has been extensively studied, and has been shown to be controlled by numerous transcription faαors that recognize specific DNA sequences in the Ig enhancers (Kadesch, T., Immunol. Today 13: 31-36 (1992)). Recent reports on E2A null mutant mice that lack mature B cells clearly depiα the impaα of these bHLH proteins on B cell development (Bain, G. et al., Cell 79: 885-892 (1994); Zhuang, Y. et al., Cell 79: 875-884 (1994)). Genetic analysis of neural cell fate and sex determination in Drosophila provided in vivo evidence for interaαion between bHLH proteins (reviewed in Jan, Y.N. et al., Cell 75: 827-830 (1993)). For instance, bHLH proteins encoded by daughterless (da) and the achaete-scute complex (AS-C) heterodimerize and positively regulate sensory organ formation. On the other hand, the genes encoding negative regulators such as hairy and extramacrochaetae are required to control the appropriate pattern of neural precursor distribution. Moreover, because cell differentiation is often associated with the suppression of proliferation, some HLH proteins have also been implicated in the regulation of cell growth. One of the most extensively smdied may be Myc, a bHLH protein encoded by the c-myc oncogene (reviewed in Marcu, K. B. et al., Annu. Rev. Biochem. 61: 809-860 (1992)). The negative regulator Id proteins which inhibit differentiation by forming inactive heterodimers with bHLH proteins, thereby may be required for proliferation. For example, the level of Id expression is higher in undifferentiated proliferating cells (Benezra, R. et al. (1990) Cell 61: 49-59). Also, antisense oligonucleotide against Id mRNA inhibits re-entry to the cell cycle (Barone, M.V. et al., P.N.A.S. (USA) 91: 4985-4988 (1994); Hara, E. et al., J. Biol. Chem. 269: 2139-2145 (1994)), and cell cycle progression is accelerated in Id2 stable transfectant cell lines (Ivarone, A. et al. (1994) Genes & Dev 8: 1270-1284). The following materials and methods were used in mis example.
Cell Culture and Reagents Human T cells were prepared as described previously (Beadling, C. et al., P.N.A.S.
(USA) 90: 2719-2723 (1993)); in short, peripheral blood mononuclear cells were cultured in RPMI 1640 supplemented with 10% (v/v) heat-inaαivated fetal calf serum (FCS) and antibiotics in the presence of OKT3 (Ortho Pharmaceuticals) for 3 days, then for an additional 11 days in the presence of IL-2 (Takeda Chemical). The cells were subsequently removed from of IL-2 for 36 hr, followed by a 12 hr stimulation with phorbol-12, 13-dibutyrate (Sigma) to augment the expression of high-affinity IL-2 receptor. Such treatment enabled the generation of a G0/G1- synchronized cell population, comprised of >90 % CD8+ T lymphocytes (Gullberg, M. et al., J. Exp. Med. 163: 270-284 (1986)). Kit 225 is an IL-2-dependent human T cell line (Hori, T. et al., Blood 70: 1069-1072 (1987)). Ba/F3 and CTLL2 are mouse cell lines dependent on IL-3 and IL-2, respeαively. Both Kit 225 and CTLL2 were maintained in RPMI 1640 supplemented with 10 % (v/v) FCS and 500 pM recombinant human IL-2. Ba/F3 was maintained in RPMI 1640 supplemented with 10 % (v/v) FCS and 5 % (v/v) conditioned medium from fibroblasts transfeαed with mouse IL-3 as a source of IL-3. Recombinant mouse IL-3 was purchased from Genzyme. Before using for experiments, cell lines were made quiescent by growth faαor deprivation for 72 hr for Kit 225, 12 hr for Ba/F3 and 2 hr for CTLL2. Forskolin was obtained from Sigma. Human interferon (IFN) β was from GIBCO BRL. Proliferation was monitored by measuring the incorporation of [3H-methyl]thymidine (Amersham) into ten thousand cells incubated with indicated reagents in 200 fl for 24 hr at 37 °C. The culture was pulsed with 0.5 fCi [3H]thymidine for the last 4 hr prior to harvest.
Northern Hybridization
Total cellular RNA was isolated by RNAzolB (Tel-Test) and fraαionated on a 1.2 % agarose formaldehyde gel. RNA was visualized with ethidium bromide. After eleαrophoresis, RNA was transferred and fixed to Hybond-N+ nylon membrane (Amersham) with 40 mM NaOH. Multiple Tissue Northern Blot membranes were purchased from Clontech. Membranes were hybridized with the radiolabeled probe for 3 hr to overnight at 65 °C in Rapid-Hyb hybridization solution (Amersham), washed twice with 2 x SSC/0.1 % SDS (1 x SSC = 150 mM NaCl/15 mM sodium citrate, pH 7.0) at room temperature for 15 min, once with 0.5 x SSC/0.1 % SDS at 60°C for 15 min and subjected to autoradiography.
cDNA Library Screening
The λgtlO cDNA libraries were construαed and screened according to the standard molecular biology procedure (Sambrook, J et al., Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)). Poly(A)+ RNA was isolated from IL-2-stimulated normal human T cells prepared as above and cDNA synthesis was primed with both oligo(dT)12-18 and random hexamers. The recombinant phages were screened with radiolabeled CR8 insert. For mouse CR8, a λgtlO cDNA library from IL-2-stimulated mouse splenocytes were screened wid human CR8 insert under low-stringency condition.
Sequence Analysis CR8 cDNA sequence was analyzed by the fluorescence-based dideoxynucleotide termination method (Taq DyeDeoxyTM Terminator Cycle Sequencing Kit, Perkin Elmer) on the Applied Biosystems Model 373A DNA sequencer. Consensus sequences were constructed and analyzed with the help of the University of Wisconsin GCG software package. The BLAST algorithm from the National Center for Biotechnology Information (NCBI) was also employed for nucleotide and amino acid sequence homology search (Altschul, S. F. et al. (1990) J. Moi. Biol. 215: 403-410). Preparation of Recombinant Proteins
The recombinant CR8 protein with histidine-tag was prepared using the XpressTM System (Invitrogen) according to the manufacturer's protocol. cDNA corresponding to the CR8 bHLH domain was obtained by PCR. The sequences of the primers, 5'- GGGGTCTACCAGGGATGTAC-3' (SEQ. ID NO: 15) for the 5' side, and 5'- GTAAACCACTCTGCAGGGCAATGA-3' (SEQ. ID NO: 16) for the 3' side, were slightly different from me final consensus sequence for CR8, but the difference did not affeα the core bHLH motif. The PCR product was cloned into pT7Blue T-veαor (Novagen) and subsequently into pRSET-A veαor at BamHI and Hindlll sites. Construαs were confirmed by DNA sequencing. The protein was overexpressed in JM109 at 37°C in the presence of isopropylthio-b- D-galaαoside (IPTG) by infeαing the baαeria with M13 phages that contain the T7 RNA polymerase gene. The cells were lysed witii 100 fg/ml lysozyme in native binding buffer (20 mM sodium phosphate, pH 7.8, 500 mM NaCl), the lysate was loaded on a ProBondTM Ni2+ column, and the recombinant protein was eluted with native-imidazole elution buffer (20 mM sodium phosphate, pH 6.0, 500 mM NaCl, 500 mM imidazole). The protein was then dialyzed against lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 % glycerol, 0.1 % Triton X-100, 1 mM DTT) and concentrated on a Microcon 10 (Amicon). To confirm the purity and the quantity, the protein was fraαionated on a 12% SDS-polyacrylamide gel with protein standards of known concentrations and visualized by silver staining. The recombinant protein corresponding to the bHLH domain of da was supplied by Dr. Michael Caudy (Cornell University Medical College).
Mobility Shift Assay
The oligonucleotide probes used for the electrophoretic mobility shift assay (EMSA) are as follows: the Class A site used was the CACCTG hexamer (CAGGTG for the opposite strand) from the T5 promoter region of the Drosophila AS-C (Villares, R. et al., Cell 550: 415-424 (1987)) (5'-GATCGTAGTCACGCAGGTGGGATCCCTA-3' (SEQ. ID No.: 17) and 5'- GATCTAGGGATCCCACCTGCGTGACTAC-3' (SEQ. ID No.: 18) for the opposite strand), the Class B site was the CACGTG hexamer from the USF binding site in the adenovirus major late promoter (Gregor, P. D. et al. (1990) Genes & Dev. 4:1730-1740) (5'- GATCGGTGTAGGCCACGTGACCGGGTGT-3' (SEQ. ID No.: 19) and 5'- GATCACACCCGGTCACGTGGCCTACACC-3') (SEQ. ID No.: 20), the Class C site was the CACGCG hexamer (CGCGTG for the opposite strand) from the AS-C T5 promoter (5'- GATCGGCAGCCGGCACGCGACAGGGCC-3' (SEQ. ID No.: 21) and 5'- GATCGGCCCTGTCGCGTGCCGGCTGCC-3') (SEQ. ID No.: 22), and the N-box (CACNAG) was the double hexamer sequence from the Enhancer of split [E(spl)] m8 promoter (Klimbt, C. et al, EMBO J. 8:203-210. (1989) (5'-GATCACGCCACGAGCCACAAGGATTG-3' (SEQ. ID No.: 23) and 5'-GATCCAATCCTTGTGGCTCGTGGCGT-3' (SEQ. ID No.: 24)). One strand of the oligonucleotide was labeled with [g-32P]ATP by T4 polynucleotide kinase, hybridized with three times excess of the opposite strand, and purified using MERmaid oligonucleotide purification kit (BIO 101). 150 ng of the protein was allowed to bind to 50,000 cpm (equivalent to 0.5 ng in a typical experiment) of the labeled probe for 15 min at room temperature in 20 mM Hepes, pH 7.6, 50 mM KC1, 10 mM DTT, 5 % glycerol, 0.5 mM EDTA and 0.3 mg/ml BSA. Two microgram of poly(dl-dC) was added to each 20 fl reaαion as on-specific DNA. Samples were analyzed on a 5% native polyacrylamide gel and visualized by autoradiography.
Regulation of CR8
CR genes were originally defined in IL-2 stimulated normal human T cells. To examine CR8 expression in cytokine-dependent cell lines, the level of CR8 expression was measured by Northern hybridization in the IL-2-dependent human T cell line Kit 225, the IL-3-dependent mouse pro-B cell line Ba/F3, and me IL-2-dependent mouse T cell line CTLL2. A single 3.2 Kb species hybridized to the cDNA probe, and in all three cell lines tested, the level of CR8 was clearly augmented when the cells were stimulated with their respeαive growth faαors. Correlation between die level of CR8 and that of DNA synthesis was in the presence of growth- inhibitory agents was also examined. In this regard, increases in cytoplasmic cAMP are known to inhibit the growth of many cell types, including lymphocytes (Johnson, K. W. et al., P.N.A.S. (USA) 85: 6072-6076 (1988)). IFNs also exert antiproliferative aαivity on many cell types (Pestka, S. et al., ) Annu. Rev. Biochem. 56: 727-777 (1987)). Therefore, Kit 225 was stimulated with IL-2, IFNβ, or forskolin, which increases cytoplasmic cAMP by aαivating adenylate cyclase, either alone or in combination. IL-2-dependent [3H]thymidine incorporation was inhibited by IFNb and forskolin in Kit 225 cells in a dose-dependent fashion. IFNβ and forskolin inhibit IL-2-dependent [3H]thymidine incorporation by Kit 225 cells. While forskolin was capable of reducing the IL-2-dependent [3H]thymidine incorporation almost to the basal level, IFNβ- mediated inhibition never exceeded 70% of the maximal incorporation in several independent experiments. The expression of CR8 was compared with that of c-myc, an IL-2-inducible immediate-early gene that encodes a bHLH protein and is implicated for cell proliferation (Marcu, K. B. et al. (1992) Annu. Rev. Biochem. 61: 809-860). CR8 transcripts were moderately induced, not only by IL-2, but also by IFNβ or forskolin alone. Furthermore, the simultaneous stimulation of quiescent Kit 225 cells with IL-2 and IFNβ, or IL-2 and forskolin, did not suppress the IL-2-induced expression of CR8 transcripts. In contrast, IL-2-induαion of c-myc expression was substantially inhibited in die presence of forskolin, while IFNβ did not significantly reduce IL-2-promoted c-myc expression.
Cloning of CR8
The original human CR8 clone isolated from the thiol-selected library had a 1.5 Kb insert, while the full-length mRNA transcript was estimated to be 3.2 Kb from Northern blotting experiments. As the CR8 clone did not have a long open reading frame, two full-length cDNA clones of human CR8 were isolated from a λgtlO human T cell cDNA library after two rounds of screening with cDNA fragments of the CR8 clone. These two clones were fully sequenced on both strands and the coding strand sequence determined (SEQ. ID No.: 13), and the amino acid sequence was deduced (SEQ. ID No.: 14). When the final consensus cDNA sequence of 2970 bp (excluding the poly(A) stretch) was screened against the nonredundant nucleotide databases using the NCBI BLAST E-mail server (GenBank release 86.0), no known genes in the database shared significant homology with CR8 except for nine EST sequences (Adams, M. D. et al. (1991) Science 252: 1651-1656). CR8 has an open reading frame of 412 amino acids (SEQ. ID No.: 14), with an in-frame termination codon at position 198 followed by Met at position 240 in a reasonable context for translation initiation (CGCCATGG) (Kozak, M., Cell 44: 283-292 (1986)). The MOTIFS program in the GCG package prediαed the presence of an HLH motif in CR8.
A mouse CR8 cDNA fragment corresponding to nt 388 to 2720 of die human sequence was also isolated from a λgtlO mouse cDNA library by comparison of CR8 with other bHLH Proteins. The protein database search with the putative peptide sequence revealed that CR8 shares homology with the bHLH proteins encoded by Drosophila hairy gene and the enhancer of split complex [E(spl)-C] of neurogenic genes. Figures 1 A and IB show a sequence comparison of CR8 and other HLH proteins. Protein alignments were made to maximize homology within the bHLH domain. Amino acids conserved among most HLH proteins are shaded. The proline residues in the basic region and the arginine residues at position 13 ("R13") are boxed. The boxed alanine residue in MyoD is the one whose substitution to proline abrogated the DNA binding and muscle-specific gene aαivation aαivity of MyoD (Davis, R. L. et al. (1990) Cell 60: 733-746). [h], human; [D], Drosophila melanogaster; [r], rat; and [m], mouse. Sources for sequences: hairy, (Rushlow, C. A. et al., EMBO J. 8: 3095-3103 (1989)); Enhancer of split [E(spl)]m7, (Klimbt, C. et al., EMBO J. 8: 203-210 (1989)); deadpan (dpn), (Bier, E. et al., Genes & Dev 6: 2137-2151 (1992)); HES-1, (Sasai, Y. et al.. Genes & Dev 6: 2620-2634 (1992)); daughterless (da), (Caudy, M. et al.. Cell 55: 1061-1067 (1988)); E12 and E47, (Murre, C. et al., Cell 56: 777-783 (1989)); MyoD, (Davis, R. L. et al.. Cell 51: 987-1000 (1988)); Tal-1, (Begley, C. G. et al., P.N.A.S. (USA) 86: 10128-10132 (1989)); USF, (Gregor, P. D. et al.. Genes & Dev 4: 1730-1740 (1990)); Max, (Blackwood, E. M. et al., Science 251: 1211-1217 (1991)); N-myc, (Slamon, D. J. et al., Science 232: 768-772 (1986)); L-myc, (Kaye, F. et al., Moi. Cell. Biol. 8: 186-195 (1986)); c-myc, (Gazin, C. et al., EMBO J. 3: 383-387(1984)); extramacrochaetae (emc), (Ellis, H.M. et al., Cell 61: 27-38 (1990); Garrell, J. et al., Cell 61: 39-48 (1990)) and Idl, (Benezra, R. et al., Cell 61: 49-59 (1990)).
When CR8 was aligned with other bHLH proteins (Figures 1A and IB), it was clear that most of the residues conserved throughout the family were present in CR8. Taken together with the result of the MOTIFS program, it was concluded that CR8 is a bHLH protein. The amino acid sequence of the 58-residue bHLH domain of CR8 showed 40% identity to hairy, 41 % to E(spl)m7, and 45% to one of their mammalian counterparts HES-1. This degree of amino acid identity accounts well for the failure to detect any significant homology to any known bHLH proteins at the nucleotide sequence level. The amino acid sequence for human and mouse CR8 was 100% identical in the bHLH domain.
Figure IB shows a sequence comparison of CR8 and hairy-related bHLH. Conserved amino acids are shaded. Note that HES-2, 3 and 5 proteins do not align perfectly in the hairy- related homology region (HRHR)-2. Sources for sequences: HES-2, (Ishibashi, M. et al., Eur. J. Biochem. 215: 645-652 (1993)); HES-3, (Sasai, Y. et al., Genes & Dev 6: 2620-2634 (1992)); HES-5, (Akazawa, C. et al., J. Biol. Chem. 267: 21879-21885 (1992)); human hairy-like (HHL), (Feder, J. N. et al., Genomics 20: 56-61 (1994)); Drosophila melanogaster hairy [h(m)], (Rushlow, CA. et al., EMBO J. 8: 3095-3103 (1989)); Drosophila virilis hairy [h(v)], (Wainwright, S. M. et al., Moi. Cell. Biol. 12:2475-2483 (1992)); Tribolium hairy [h(T)]t (Sommer, R. J. et al., Nature 361: 448-450 (1993)); E(spl)m5 and m8, (Klimbt, C. et al., EMBO J. 8: 203-210 (1989)); E(spl)m3, b/A, g/B, and d/C, (Deldakis, C. et al., P.N.A.S. (USA) 89: 8731-8735 (1992); Knust, E. et al., Genetics 132: 505-518 (1992)). As shown in Figure 15B, the amino acid sequence of the bHLH region of CR8 is aligned with hairy, bHLH proteins of the E(spl)-C, deadpan (dpn) and their mammalian homologs (the term "hairy-related bHLH proteins" refer to them colleαively). Among all the bHLH proteins described thus far, CR8 is the only one with a proline residue in the basic region, other than the hairy-related bHLH proteins. However, while the position of the proline residue is strictly conserved throughout the hairy-related bHLH proteins, in CR8 it is offset N-terminally by two residues. CR8 and hairy-related bHLH proteins are different in the C-terminus as well; all the hairy-related bHLH proteins terminate with a specific Trp-Arg-Pro-Trp (WRPW) motif, which is absent in CR8. Nevertheless, CR8 showed appreciable homology to other hairy-related bHLH proteins in die region immediately C-terminal to me bHLH domain, which has been shown previously to be rich in hydrophobic residues, and proposed to form two more α-helices in bHLH proteins of the E(spl)-C (43). This region is referred to herein as the "hairy-related homology region (HRHR)-2", the HRHR-1 being the bHLH domain. The region N-terminal to the bHLH domain and the C-terminal half of the CR8 protein are rich in proline (8 proline residues between positions 1 and 30, 22 between 310 and 405). Notably, there are no known proteins in the data bases that share homologies to these most N-terminal and C-terminal regions of CR8.
Tissue distribution of CR8 transcripts
Murre et al. (1989) Cell 58: 537-544, categorized bHLH proteins based upon tiieir tissue distribution. While proteins such as MyoD and AS-C gene produαs show a cell-type specific expression, others such as E12/E47 and da are fairly ubiquitously expressed. The tissue distribution of CR8 was analyzed using a Multiple Tissue Northern blot. CR8 transcripts of the expected size (3.2 Kb) were deteαed in all tissues examined except placenta. This included heart, brain, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood leukocytes. The expression of CR8 in peripheral blood leukocytes was unexpeαed, in that CR8 is not expressed by quiescent T cells. This may reflect much higher sensitivity of Multiple Tissue Northern blot prepared from poly(A)+ RNA compared to our previous Northern blots, which used total RNA. Alternatively, the contribution of other leukocytes such as B cells, NK cells, monocytes and granulocytes that were not present in the original T cell preparations could account for CR8 expression by the peripheral blood leukocytes.
DNA-binding activity of CR8
The canonical bHLH binding sequence is called d e E-box, CANNTG, originally identified in the immunoglobulin heavy chain enhancer (Ephrussi, A. et al., Science 227: 134-140 (1985)). Many bHLH proteins were later divided into two mutually exclusive classes, depending on whether they bind to me Class A sites (CAGCTG/CACCTG) or the Class B sites (CACGTG/CATGTG) (Dang, C.V. et al., P.N.A.S. (USA) 89: 599-602 (1992)). The presence of an arginine residue at position 13 ("R13") in the basic region, which CR8 contains, is considered to be the key structural criterion that defines Class B binding specificity. However, despite the presence of "R13", hairy-related bHLH proteins are reported to prefer non-canonical binding sites such as the N-box (CACNAG) (Akazawa, C. et al., J. Biol. Chem. 267: 21879-21885 (1992); Sasai, Y. et al., Genes & Dev 6: 2620-2634 (1992); Tietze, K. et al., P.N.A.S. (USA) 89: 6152- 6156 (1992)) or the Class C (CACGCG) sites (Ohsako, S. et al., Genes & Dev 8: 2743-2755 (1994); Van Doren, M. et al., Genes & Dev 8: 2729-2742 (1994)). Therefore, the binding of CR8 to all of these sites was tested. Since it is well documented mat the bHLH domain is sufficient to determine its DNA binding specificity (Pognonec, P. et al., Moi. Cell. Biol. 11: 5125-5136 (1994)), the bHLH domain of CR8 (CR8 bHLH) expressed in E. coli was employed for this study. A histidine-tag was added to facilitate the purification of the recombinant protein. While most of the recombinant protein localized in inclusion bodies, there was still enough soluble protein in the cytoplasm, thereby enabling its purification under native conditions using a Ni2 + column. A single band of protein was deteαed at the expeαed size (16.6 kD with the histidine-tag) by silver staining. EMSA was carried out using this recombinant protein.
CR8 bHLH protein strongly binds to the Class B (CACGTG) and the Class C (CACGCG) sites, and weakly to the N box (CACNAG) sequence but not to the Class A (CACCTG) site. To examine the relative binding affinity, a large excess of non-labeled oligonucleotide was added to the reaαion as competitor, the binding of CR8 bHLH to the radiolabeled Class B site can be abolished partially by a 50-fold excess, and completely by a 500-fold excess of Class B site, while a 500-fold excess of Class C site only partially displaced CR8 bHLH from the labeled Class B probe, and the N-box sequence did not affect the binding at all. Thus, since all these experiments were done in die absence of other HLH proteins, it appears that CR8 bHLH bound to die Class B sequence as a homodimer witii the highest affinity.
The CR8 gene encodes a novel bHLH protein diat appears to fit into a class by itself. Other than c-myc, CR8 is the first bHLH -containing protein found to be induced by cytokines. Also, from its prediαed amino acid sequence, CR8 clearly contains a bHLH motif most closely related to me hairy family, but the amino acid sequence of the basic region differs from other hairy-related proteins: the position of the proline residue is N-terminal to the defining proline of me hairy-related proteins, and CR8 lacks the C-terminal WRPW sequence found in all other hairy-related-related proteins. These differences in the amino acid sequence, especially of the basic region, most likely account for the unique binding specificity of the CR8 bHLH domain. Instead of preferring Class C sites according to the other hairy-related family members (Ohsako, S. et al., Genes & Dev 8: 2743-2755 (1994); Van Doren, M. et al., Genes & Dev 8: 2729-2742 (1994)), CR8 binds preferentially to Class B sites. The identification of CR8 as a bHLH protein, thereby functioning, most likely, as a regulator of subsequent gene expression stimulated by IL-2, provides a link between the immediate biochemical events triggered by cytokine receptors and the subsequent events of proliferation and/or differentiation. Thus far, IL-2 has been found to aαivate the serine/threonine kinase proto-oncogene Raf-1 (Turner, B. et al., P.N.A.S. (USA) 88: 1227- 1231 (1991); Zmuidzinas, A. et al., Moi. Cell Biol. 11: 2794-2803 (1991)) and the tyrosine- specific kinases JAK 1 and JAK 3 (Beadling, C. et al., EMBO J. 13: 5605-5615 (1994); Miyazaki, T. et al., Science 266: 1045-1047 (1994); Russell, S.M. et al., Science 266: 1042-1045 (1994)).
From the results described herein comparing the effects of IFNa and forskolin on CR8 and c-myc gene expression, the regulation of these two bHLH genes is clearly distinα. It is also of interest that although IFNβ antagonizes IL-2-promoted cell cycle progression, it promotes the expression of both CR8 and c-myc. Indeed, induction of c-myc by IFNβ was unexpeαed, as it was previously reported to be suppressed by IFNs (Einat, M. et al., Nature 313: 597-600 (1985)). The bHLH region of CR8 is most homologous to that of hairy and the bHLH proteins of the E(spl)-C. In Drosophila, the hairy-related bHLH proteins funαion as transcriptional repressors, and this aαivity requires the basic DNA binding region, as well as the interaαion with a non- HLH protein termed groucho (gro) via the C-terminal WRPW motif (Paroush, Z. et al. (1994) Cell 79: 805-815). Although mammalian homologues of gro have been identified (Stifani, S. et al., Nat. Genet. 2: 119-127 (1992)), they are not likely to interaα with CR8 because CR8 lacks the WRPW motif. The results described herein indicate mat CR8 recombinant protein binds to Class B E- box sites as a homodimer. This result is consistent with the prediαions from DNA-bHLH protein co-crystals (Ferr-D'Amar, A. R. et al., EMBO J. 13: 180-189 (1994); Ferr-D'Amar, A. R. et al., Nature 363: 38-45 (1993)). However, it is noteworthy in that CR8 is the first bHLH vertebrate protein without a leucine zipper (LZ) motif found to bind Class B sites. Protein dimerization is more seleαive than DNA binding, but currently no rules are available that prediα the dimerization preference of any given HLH proteins. Even so, a Class A-binding protein seems to form DNA binding heterodimers only with other Class A proteins, and a bHLH protein with a LZ does not form heterodimers with those wimout LZs (Blackwood, E. M. et al., Science 251: 1211-1217 (1991); Prendergast, G.C. et al., Cell 65: 395-407 (1991)). Therefore, if CR8 does form heterodimers, the most likely partner is a class B-binding bHLH protein wimout a LZ. Although CR8 is most homologous to hairy in its bHLH domain, its preference for Class B E-box binding sites rather than class C sites, and its lack of a C-terminal WPRW motif, clearly sets CR8 apart and does not prediα necessarily that CR8 may aα as a transcriptional repressor as do hairy- related proteins. Recently, Id proteins that lack a basic region have been shown to favor proliferation, presumably by forming heterodimers with differentiation inducing bHLH proteins, diereby preventing DNA binding and transcriptional aαivation of genes that program differentiation (Barone, M. V. et al. (1994) Proc. Nad. Acad. Sci. USA 91: 4985-4988; Hara, E. et al. (1994) J. Biol. Chem. 269: 2139-2145; Iavarone, A. et al. (1994) Genes & Dev 1270- 1284). Therefore, CR8 could promote proliferation by suppressing differentiation by either of these transcriptional repressor mechanisms. Alternatively, CR8 could also aαivate transcription like the bHLH-LZ Myc family.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS: 1. A composition comprising a substantially pure polypeptide which comprises an amino acid sequence seleαed from the group consisting of CRI, CR2, CR3, CR5, CR6, CR8, homologues thereof and of CR4 and CR7 having at least about 60% homology thereto, fragments thereof, and combinations thereof. 2. The composition of claim 1, wherein the polypeptide comprises the amino acid SEQ. ID No: 2. 3. The composition of claim 1, wherein the polypeptide comprises the amino acid SEQ. ID No: 4. 4. The composition of claim 1, wherein the polypeptide comprises the amino acid SEQ. ID No: 6. 5. The composition of claim 1, wherein the polypeptide comprises the amino acid SEQ. ID No: 10. 6. The composition of claim 1, wherein the polypeptide comprises the amino acid SEQ. ID No: 12. 7. The composition of claim 1, wherein the polypeptide comprises the amino acid SEQ. ID No: 14. 8. The composition of claim 1, further comprising a diluent or carrier. 9. The composition of claim 8, wherein the carrier or diluent comprises a pharmaceutically acceptable carrier or diluent. 10. A composition comprising a fusion polypeptide which comprises the polypeptide of claim 1 , and an additional polypeptide comprising an amino acid sequence unrelated to at least one of the polypeptides. 11. The composition of claim 10, further comprising a diluent or carrier. 12. A substantially pure polynucleotide, which comprises a nucleic acid sequence encoding the polypeptide of claim 1. 13. The polynucleotide of claim 12, which comprises the antisense nucleic acid sequence. 14. The polynucleotide of claim 12, which comprises at least one nucleic acids encoding a polypeptide selected from the group consisting of SEQ. ID No: 2, SEQ. ID No: 4, SEQ. ID No: 6, SEQ. ID No: 10, SEQ. ID No: 12, SEQ. ID No: 14, polypeptides having at least about 60% homology thereto and to SEQ. ID No: 8 and SEQ. ID No: 28, and combinations thereof. 15. The polynucleotide of claim 14, which comprises a nucleic acid sequence seleαed from the group consisting of SEQ. ID No: 29, SEQ. ID No: 30, SEQ. ID No: 31, SEQ. ID No: 32, SEQ ID No: 33, SEQ. ID No: 34, and nucleic acids having at least about 60% homology thereto and to SEQ. ID No: 7 and SEQ. ID No:27, and combinations thereof. 16. The polynucleotide of claim 15, which comprises at least ten consecutive nucleic acids thereof. 17. The polynucleotide of claim 12, which comprises at least about 10 nucleic acids from SEQ. ID No: 29. 18. The polynucleotide of claim 12, which comprises at least about 10 nucleic acids from SEQ. ID No: 30. 19. The polynucleotide of claim 12, which comprises at least about 10 nucleic acids from SEQ. ID No: 31. 20. The polynucleotide of claim 12, which comprises at least about 10 nucleic acids from SEQ. ID No: 32. 21. The polynucleotide of claim 12, which comprises at least about 10 nucleic acids from SEQ. ID No: 33. 22. The polynucleotide of claim 12, which comprises at least about 10 nucleic acids from SEQ. ID No: 34. 23. A probe/primer comprising a nucleic acid which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence of the polynucleotide of claim 12. 24. A composition, comprising the polynucleotide of claim 12, and a diluent or carrier. 25. A composition comprising a veαor which has a nucleic acid encoding the polypeptide of claim 1, linked in reading frame thereto. 26. A host cell transfeαed with the veαor of claim 25. 27. A method for producing a recombinant polypeptide, comprising culturing the cell of claim 26, in an expression medium under conditions effeαive to express the polypeptide; and allowing me polypeptide to accumulate. 28. The method of claim 27, further comprising separating the polypeptide from the medium and the cells. 29. A transgenic non-human animal, comprising a trans-gene encoding the polypeptide of claim 1. 30. The animal of claim 29, comprising at least one allele encoding a polypeptide comprising a mutated or mis-expressed amino acid sequence seleαed from the group consisting of SEQ. ID NO: 2, SEQ. ID No: 4, SEQ. ID No: 6, SEQ. ID No: 8, SEQ. ID No: 10, SEQ. ID No: 12, SEQ. ID No: 14, SEQ. ID No: 28, and combinations thereof. 31. A composition comprising an antibody which seleαively binds the polypeptide of claim 1. 32. The composition of claim 31, wherein the antibody comprises a monoclonal antibody. 33. The composition of claim 31, further comprising a diluent or carrier. 34. The composition of claim 33, where the diluent or carrier comprises a pharmaceutically acceptable diluent or carrier.
PCT/US1996/009194 1995-06-05 1996-06-05 Ligand-stimulated gene expression WO1996039427A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP96921319A EP0832117A1 (en) 1995-06-05 1996-06-05 Ligand-stimulated gene expression

Applications Claiming Priority (12)

Application Number Priority Date Filing Date Title
US08/463,081 1995-06-05
US08/465,585 1995-06-05
US08/462,390 1995-06-05
US08/465,585 US6027914A (en) 1991-11-20 1995-06-05 Nucleic acids encoding CR6 polypeptide vector and transformed cell thereof, and expression thereof
US08/461,379 US5871961A (en) 1991-11-20 1995-06-05 Nucleic acids encoding CR2 polypeptides, vector and transformed cell thereof, and expression thereof
US08/462,390 US5882894A (en) 1991-11-20 1995-06-05 Nucleic acids encoding CR8 polypeptides, vector and transformed cell thereof, and expression thereof
US08/462,337 1995-06-05
US08/462,337 US6051398A (en) 1991-11-20 1995-06-05 Nucleic acids encoding CR3 polypeptide, vector and transformed cell thereof, and expression thereof
US08/461,379 1995-06-05
US08/463,081 US5871960A (en) 1991-11-20 1995-06-05 Nucleic acids encoding CR5 polypeptide, vector and transformed cell thereof, and expression thereof
US08/463,074 US6020155A (en) 1991-11-20 1995-06-05 Nucleic acids encoding CR1 fusion protein, vector, transfected cell and expression
US08/463,074 1995-06-05

Publications (1)

Publication Number Publication Date
WO1996039427A1 true WO1996039427A1 (en) 1996-12-12

Family

ID=27560022

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/009194 WO1996039427A1 (en) 1995-06-05 1996-06-05 Ligand-stimulated gene expression

Country Status (2)

Country Link
EP (1) EP0832117A1 (en)
WO (1) WO1996039427A1 (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0787798A2 (en) * 1996-02-09 1997-08-06 Otsuka Pharmaceutical Co., Ltd. GRP17 gene
WO1998020023A1 (en) * 1996-11-01 1998-05-14 The Walter And Eliza Hall Institute Of Medical Research Therapeutic and diagnostic agents capable of modulating cellular responsiveness to cytokines
US5846780A (en) * 1996-10-04 1998-12-08 Millennium Pharmaceuticals, Inc. Murine RATH gene
WO1999002677A1 (en) * 1997-07-11 1999-01-21 Chugai Seiyaku Kabushiki Kaisha Gene originating in human chondrocyte
US6020142A (en) * 1996-10-04 2000-02-01 Millennium Pharmaceuticals, Inc. Rath genes and polypeptides and methods for the treatment and diagnosis of immune disorders
WO2000036147A1 (en) * 1998-12-12 2000-06-22 Samyang Genex Corporation Cancer diagnosis method using cell growth inhibiting and cell differentiation specific syg972 gene and genomic dna and promoter thereof
WO2001035732A1 (en) * 1999-11-16 2001-05-25 The Walter And Eliza Hall Institute Of Medical Research An animal model for studying hormone signalling and method of modulating the signalling
WO2003106681A2 (en) * 2002-06-14 2003-12-24 Grünenthal GmbH Antisense oligonucleotides against pim1

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CELL, 21 August 1992, Vol. 70, DICKINSON et al., "A Tissue-Specific MAR/SAR DNA-Binding Protein with Unusual Binding Site Recognition", pages 631-645. *
DNA AND CELL BIOLOGY, 1994, Vol. 13, No. 2, SIDEROVSKI et al., "A Human Gene Encoding a Putative Basic Helix-Loop-Helix Phosphoprotein Whose mRNA Increases Rapidly in Cycloheximide-Treated Blood Mononuclear Cells", pages 125-147. *
MOLECULAR PHARMACOLOGY, 1994, Vol. 46, REGAN et al., "Cloning of a Novel Human Prostaglandin Receptor with Characteristics of the Pharmacologically Defined EP2 Subtype", pages 213-220. *
ONCOGENE, June 1991, Vol. 6, ABDOLLAHI et al., "Sequence and Expression of a cDNA Encoding MyD118: a Novel Myeloid Differentiation Primary Response Gene Induced by Multiple Cytokines", pages 165-167. *
THE EMBO JOURNAL, November 1995, Vol. 14, No. 12, YOSHIMURA et al., "A Novel Cytokine-Inducible Gene CIS Encodes an SH2-Containing Protein that Binds to Tyrosine-Phosphorylated Interleukin 3 and Erythropoietin Receptors", pages 2816-2826. *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0787798A2 (en) * 1996-02-09 1997-08-06 Otsuka Pharmaceutical Co., Ltd. GRP17 gene
EP0787798A3 (en) * 1996-02-09 2000-01-05 Otsuka Pharmaceutical Co., Ltd. GRP17 gene
US6146827A (en) * 1996-10-04 2000-11-14 Millennium Pharmaceuticals, Inc. Rath genes and polypeptides and methods for the treatment and diagnosis of immune disorders
US5846780A (en) * 1996-10-04 1998-12-08 Millennium Pharmaceuticals, Inc. Murine RATH gene
US6020142A (en) * 1996-10-04 2000-02-01 Millennium Pharmaceuticals, Inc. Rath genes and polypeptides and methods for the treatment and diagnosis of immune disorders
WO1998020023A1 (en) * 1996-11-01 1998-05-14 The Walter And Eliza Hall Institute Of Medical Research Therapeutic and diagnostic agents capable of modulating cellular responsiveness to cytokines
GB2331753A (en) * 1996-11-01 1999-06-02 Inst Medical W & E Hall Therapeutic and diagnostic agents capable of modulating cellular responsiveness to cytokines
WO1999002677A1 (en) * 1997-07-11 1999-01-21 Chugai Seiyaku Kabushiki Kaisha Gene originating in human chondrocyte
US6392022B1 (en) 1997-07-11 2002-05-21 Chugai Seiyaku Kabushiki Kaisha Gene originating in human chondrocyte
WO2000036147A1 (en) * 1998-12-12 2000-06-22 Samyang Genex Corporation Cancer diagnosis method using cell growth inhibiting and cell differentiation specific syg972 gene and genomic dna and promoter thereof
US6573371B2 (en) 1998-12-12 2003-06-03 Samyang Genex Corporation Cell growth inhibiting and cell differentiation specific SYG972 gene, genomic DNA and promoter thereof
WO2001035732A1 (en) * 1999-11-16 2001-05-25 The Walter And Eliza Hall Institute Of Medical Research An animal model for studying hormone signalling and method of modulating the signalling
WO2003106681A2 (en) * 2002-06-14 2003-12-24 Grünenthal GmbH Antisense oligonucleotides against pim1
WO2003106681A3 (en) * 2002-06-14 2004-07-15 Gruenenthal Gmbh Antisense oligonucleotides against pim1

Also Published As

Publication number Publication date
EP0832117A1 (en) 1998-04-01

Similar Documents

Publication Publication Date Title
US6100031A (en) Methods for diagnosis of colon cancer by detecting Roch083 mRNA
Neckelmann et al. The human ATP synthase β subunit gene: sequence analysis, chromosome assignment, and differential expression
WO1998012302A1 (en) Weight control pathway genes and uses therefor
US6057427A (en) Antibody to cytokine response gene 2(CR2) polypeptide
JP2002525067A (en) Leptin-inducible gene
US6143491A (en) Therapeutic compositions and methods and diagnostic assays for type II diabetes involving HNF-1
WO1998004590A1 (en) Conservin compositions and therapeutic and diagnostic uses therefor
IL182749A (en) Vwfa and/or ant_ig domain containing proteins
US5871960A (en) Nucleic acids encoding CR5 polypeptide, vector and transformed cell thereof, and expression thereof
JP3949715B2 (en) Novel hemopoietin receptor
CN113544257A (en) Characterization and inactivation of endogenous retroviruses in chinese hamster ovary cells
WO1996039427A1 (en) Ligand-stimulated gene expression
US6436684B1 (en) Isolated human drug-metabolizing proteins, nucleic acid molecules encoding human drug-metabolizing proteins, and uses thereof
Janzen et al. ARPP-16 mRNA is up-regulated in the longissimus muscle of pigs possessing an elevated growth rate
US5871961A (en) Nucleic acids encoding CR2 polypeptides, vector and transformed cell thereof, and expression thereof
US5882894A (en) Nucleic acids encoding CR8 polypeptides, vector and transformed cell thereof, and expression thereof
US6027914A (en) Nucleic acids encoding CR6 polypeptide vector and transformed cell thereof, and expression thereof
WO1998046748A1 (en) Therapeutic compositions and diagnostic assays for diseases involving trbp
US6051398A (en) Nucleic acids encoding CR3 polypeptide, vector and transformed cell thereof, and expression thereof
US20030232336A1 (en) Novel human ion channel and transporter family members
US6130065A (en) Nitrobenzylmercaptopurineriboside (NBMPR)-insensitive, equilibrative, nucleoside transport protein, nucleic acids encoding the same and methods of use
US6020155A (en) Nucleic acids encoding CR1 fusion protein, vector, transfected cell and expression
AU6176696A (en) Interleukin-1 receptor-associated protein kinase and assays
US20030073162A1 (en) Signal peptide-containing proteins
WO1998046749A9 (en) A nitrobenzylmercaptopurineriboside (nbmpr)-insensitive, equilibrative, nucleoside transport protein, nucleic acids encoding the same and methods of use

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 1996921319

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1996921319

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: CA

WWW Wipo information: withdrawn in national office

Ref document number: 1996921319

Country of ref document: EP