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
  • C07K14/4702—Regulators; Modulating activity
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• This invention relates to nucleic acid and amino acid sequences of cullin regulators that are associated with ubiquitin ligase activity, and to methods utilizing these sequences.
• the ubiquitin-dependent proteolytic process regulates many short lived intracellular proteins, whose concentrations change promptly as the result of alterations in cellular physiological conditions. See Hochstrasser,.M. et al. (1996) Annu. Rev. Genet. 30, 405-439.; King, R.W., et al. (1996) Science 274, 1652-1659; Hershko, A. et al. (1997) Curr. Opin. Cell Biol. 9, 788-799. In addition to performing "housekeeping" functions such as homeostasis and the removal of misfolded proteins, this proteolytic process is involved in the degradation of many regulatory proteins, such as cyclins, CDK inhibitors, transcription factors, and signal transducers.
• regulatory proteins such as cyclins, CDK inhibitors, transcription factors, and signal transducers.
• ubiquitin-mediated proteolysis begins with activation of ubiquitin, a 76-amino acid protein expressed in all eukaryotic cells, in an ATP- dependent manner by an ubiquitin-activating enzyme (El or Uba).
• El ubiquitin-activating enzyme
• the activated ubiquitin forms a high energy thiolester bond with El and is passed to a cysteine residue also via a thiolester bond within an ubiquitin-conjugating enzyme designated as an E2 or Ubc.
• E2-linked ubiquitin is then transferred to a side chain amino group of a lysine residue in the substrate to form a terminal isopeptide bond, either directly or often indirectly targeted by a ubiquitin ligase known as E3.
• Substrate proteins can be linked to a single ubiquitin (monoubiquitinated) or multiple ubiquitin molecules (polyubiquitinated).
• monoubiquitinated conjugates are not clear since they do not appear to be shortlived.
• Successive covalent ligations of additional ubiquitins to the Lys 46 of the preceding ubiquitin via an isopeptide bond results in polyubiquitinated conjugates which are rapidly detected and degraded by the 26S proteosome.
• E3 is functionally, rather than structurally, defined as an ubiquitin ligase activity that is both necessary and sufficient for transfer of ubiquitin from a ubiquitin-charged E2 to a substrate, and is further believed to be involved in many polyubiquitination reactions by providing substrate specificity. Because most polyubiquitinated proteins are indiscriminately delivered to the 26S proteosome for degradation, elucidating the mechanism and regulation of E3 ligase activities has become a critical issue central to the understanding of regulated proteolysis.
• the cullin family of proteins potentially form a large number of distinct E3s as indicated by the existence of a multi-gene family and by the assembly of yeast CDC53 into at least three distinct E3 complexes: with SKP1-CDC4, with SKP1-GR.R1 and likely with SKP1-MET30 to mediate the ubiquitination of SIC 1, CLN and SWE1 proteins, respectively.
• Skowyra D., et al., (1997) Cell 91, 209-219; Feldman, R.M.R.(1997) Cell 91, 221-230; and Kaiser, P. et al, (1998) Genes & Dev. 12, 2587-2597.
• CDC53 is required for S phase entry (Mathias, N. et al., (1996) Mol. Cell Biol. 16, 6634-6643; for coupling glucose sensing to gene expression and the cell cycle (Li, F.N. and Johnston, M. (1997) EMBO J. 16, 5629-5638; and possibly for activating mitotic CLB-CDC28 activity (Kaiser, P. et al., (1998) Genes & Dev. 12, 2587- 2597).
• the C. elegans cul-1 mutant displays a hyperplasia phenotype.
• Human CUL2 is associated with the tumor suppressor VHL (von Hippel-Lindau) implicated in the regulation of the stability of hypoxia- induced mRNA (see Pause, A., et al. (1997) Proc. Natl. Acad. Sci USA. 94, 2156- 2161; Lonergan, K.M. et al., (1998) ?/. Cell Biol. 18, 732-741.
• Human CUL4A is implicated in oncogenesis by its genomic amplification and overexpression in breast cancers (Chen, L-C, et al., (1998) Cancer Res. 58, 3677-3683), and deficiency of the cullin-related APC2 results in mitotic arrest (Zachariae, W. et al., (1998) Science 279, 1216-1219; Yu, H., et al., Current Biology 6/455-466).
• E3 ubiquitin ligases The knowledge of E3 ubiquitin ligases is presently limited. Among the few characterized E3 ligases are the N-end rule ubiquitin ligase E3 ⁇ /Ubrl that recognize proteins by binding to the basic or hydrophobic residues at the amino- termini of substrate proteins (reviewed in Varshavsky, A. (1996) Proc. Natl. Acad. Sci U.S.A. 93, 12142-12149); the HECT (homologous to E6-AP carboxy terminus) domain proteins represented by the mammalian E6AP-E6 complex which functions as a ubiquitin-ligase for p53 (see Scheffher, M.
• the APC plays a crucial role in regulating the passage of cells through anaphase by promoting ubiquitin-dependent proteolysis of many proteins.
• the APC destroys the mitotic B-type cyclin for inactivation of CDC2 kinase activity and initiating cytokinesis.
• the APC is also required for degradation of other proteins for sister chromatid separation and spindle disassembly, including the anaphase inhibitors PDS1 (Cohen-Fix, 0 consult et ⁇ /.(1996) Genes & Dev. 10, 3081- 3093) and CUT2 (Funabiki, H., et al. (1996) Nature 381, 438-441), ASE1 (Juang, Y-L. et al. (1997) Science 275, 1311-1314) and the cohesion protein SCC1P
• the SCF consists of at least three subunits, SKP1, CDC53/cullin and an F-box containing protein, in which SKP1 functions as an adaptor to connect CDC53 to the F-box protein which binds directly to the substrate (Feldman, R.M.R., et al., (1997) Cell 91, 221-230; Bai, C, et al.
• CUL1 the gene cullin-1 (CUL1)
• CUL1 represents an evolutionarily conserved multigene family that includes at least seven members in C. elegans, six in humans, and three in budding yeast including Cdc53p (Kipreos, et al, supra, and Mathias, ⁇ . et al., (1996) Mol. Cell Biol. 16, 6634-6643).
• human cullin 1 directly binds to SKP1 to form a multi-subunit complex with SKP2 (an F box protein), cyclin A and CDK2 (Lisztwan, J. et al, (1998) EMBO J. 17, 368-383; Michel, J. and Xiong, Y. (1998) Cell Growth. Differ. 9, 439-445; Lyapina, S.A., et al. (1998) Proc. Natl. Acad. Sci. USA 95, 7451-7456; and Yu, Z. K. et al. (1998) Proc. Natl. Acad. Sci U. S. A.
• ROC1 and ROC2 are similar to APC11, a subunit of the APC complex.
• ROC1 and ROC2 commonly interact with all cullin proteins, while APC 11 specifically interacts with APC2.
• ROC1 functions in vivo as an essential regulator of CDK inliibitor Sicl degradation by the SCF pathway. Additionally, the inventors have found that ROC-cullin constitutes the catalytic ubiquitin ligase.
• the invention provides an isolated polynucleotide sequence encoding the protein ROC1.
• the polynucleotide sequence may be selected from the group consisting of:
• the present invention further provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences.
• the expression vector containing the polynucleotide sequence is contained within a host cell.
• the invention further provides a protein or fragment thereof encoded by a polynucleotide as given above (e.g., the protein provided herein as SEQ ID NO: 2).
• a polynucleotide as given above (e.g., the protein provided herein as SEQ ID NO: 2).
• Such proteins may be isolated and/or purified in accordance with known techniques.
• the invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a fragment thereof, the method comprising the steps of: a) culturing the host cell containing an expression vector containing at least a fragment of the polynucleotide sequence encoding ROC1 under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
• the invention also provides an antibody (e.g., a polyclonal antibody, a monoclonal antibody) which specifically binds to a protein as given above.
• the invention provides an antisense oligonucleotide complementary to a polynucleotide sequence as given above and having a length sufficient to hybridize thereto under physiological conditions.
• DNA encoding such an antisense oligonucleotide, and a nucleic acid construct having a promoter and a heterologous nucleic acid operably linked to said promoter (wherein the heterologous nucleic acid is a DNA encoding such an antisense oligonucleotide) is also an aspect of the invention.
• the invention also provides a method for detecting a polynucleotide which encodes ROC1 in a biological sample comprising the steps of: a) hybridizing the complement of the polynucleotide sequence which encodes SEQ ID NO:l to nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding ROC1 in the biological sample.
• the nucleic acid material of the biological sample is amplified by the polymerase chain reaction prior to hybridization.
• the invention provides an isolated polynucleotide sequence encoding the protein ROC2.
• the polynucleotide sequence may be selected from the group consisting of:
• ROC 1 encoded by a DNA of (a) or (b) above.
• the present invention further provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences.
• the expression vector containing the polynucleotide sequence is contained within a host cell.
• the invention further provides a protein or fragment thereof encoded by a polynucleotide as given above (e.g., the protein provided herein as SEQ ID NO: 4). Such proteins may be isolated and/or purified in accordance with known techniques.
• the invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:4, or a fragment thereof, the method comprising the steps of: a) culturing the host cell containing an expression vector containing at least a fragment of the polynucleotide sequence encoding ROC2 under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
• the invention also provides an antibody (e.g., a polyclonal antibody, a monoclonal antibody) which specifically binds to a protein as given above.
• an antibody e.g., a polyclonal antibody, a monoclonal antibody
• the invention provides an antisense oligonucleotide complementary to a polynucleotide as given above and having a length sufficient to hybridize thereto under physiological conditions.
• DNA encoding such an antisense oligonucleotide, and a nucleic acid construct having a promoter and a heterologous nucleic acid operably linked to said promoter is also an aspect of the invention.
• the invention also provides a method for detecting a polynucleotide which encodes ROC2 in a biological sample comprising the steps of: a) hybridizing the complement of the polynucleotide sequence which encodes SEQ ID NO:3 to nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding ROC2 in the biological sample.
• the nucleic acid material of the biological sample is amplified by the polymerase chain reaction prior to hybridization.
• the invention provides methods for screening bioactive agents (the term "agent” and grammatical equivalents thereof being used interchangeably with the term “compound” and the grammatical equivalents thereof) that are capable of binding to a ROC protein, wherein a ROC protein and a candidate bioactive agent are combined. The binding of the candidate bioactive agent is then determined.
• Methods of screening bioactive agents capable of interfering with the binding of ROC proteins, or of modulating the activity of a ROC protein are also aspects of the present invention.
• Such screening methods are capable of identifying compounds that have pharmacological (pharmaceutical) activity.
• Pharmaceutical formulations comprising such pharmacologically active compounds and methods of administering the same are another aspect of this invention.
• Yet another aspect of the present invention is the use of a pharmacologically active compound identified by the methods described herein for the manufacture of a medicament for the prophylactic or therapeutic use in a subject or host.
• FIGS. 1A and IB illustrate that ROC1 interacts with members of the cullin family.
• yeast HF7c cells were co- transformed with plasmids expressing indicated proteins (key) and plated onto media lacking leucine and tryptophan (-LW) to verify the presence of both bait (Leu+) and prey (Trp+) plasmids; or onto media lacking leucine, tryptophan and histidine (-LWH) to assay for interactions between bait and prey proteins.
• FIG. IB illustrates that ROCl interacts with the C-terminal portion of CULl.
• HF7c yeast cells were co-transformed with plasmids expressing indicated proteins. Protein-protein interaction was assayed as described in herein.
• FIG.2A sets forth the nucleotide sequence (SEQ ID NO:l) and the amino acid sequence (SEQ ID NO:2) of human ROCl .
• the stop codon is indicated by an asterisk.
• FIG. 2B sets forth the nucleotide sequence (SEQ ID NO: 3) and the amino acid sequence (SEQ ID NO: 4) of human ROC2.
• the stop codon is indicated by an asterisk.
• FIG. 2C illustrates the sequence comparison of ROC/ APC 11 family of proteins from representative organisms: human (Hs, Homo sapiens), -fruit fly (Dm: Drosophila melanogaster), nematodes (Ce: Caenorhabditis elegans), mouse ear cress (At: Arabidopsis thaliana), fission yeast (Sp: Schizosaccharomyces pombe), and budding yeast (Sc: Saccharomyces cerevisiae).
• FIGS 3 A, 3B and 3C illustrate the in vivo association of ROCl with cullins.
• [ 35 S]-methionine labeled lysates were prepared from HeLa cells transfected with plasmids expressing the indicated proteins. Lysates were divided into two equal amounts and immunoprecipitated with indicated antibodies and resolved by SDS-PAGE.
• FIG 3C total cell lysates prepared from HeLa cells were immunoprecipitated with indicated antibodies with (+) or without (-) competing antigen peptide. After SDS-PAGE, proteins were transferred to nitrocellulose, and analyzed by Western analysis using antibodies to CULl (lanes 1 to 4, top panel), to CUL2 (lanes 5 to 8, top panel) or to ROCl (bottom panel).
• FIG 4A-4E illustrate that ROC2 and APC11 selectively interact with cullins and APC2.
• HF7c yeast cells were co-transformed with plasmids expressing human ROC2 or human APC11 and various cullins.
• FIG. 4C and FIG. 4D illustrate the interaction between ROC2, APC11 and cullin family proteins in mammalian cells.
• HA-tagged ROC2 or APC11 were co-transfected with vectors expressing CULl or myc-tagged individual cullin proteins into HeLa cells.
• cells were pulse labeled for 2 hours with [ 5 S]-methionine.
• Cell lysates prepared from the labeled cells were divided into two equal amounts, immunoprecipitated with the indicated antibodies and resolved by SDS-PAGE.
• FIG. 4E Selective interaction between APC2 and ROC or APC 11.
• HF7c yeast cells were co-transformed with plasmids expressing indicated proteins (key). Protein-protein interaction was detennined by the yeast two-hybrid assay using selective medium lacking histidine (-LWH) supplemented with 5 mM 3-AT to suppress the low trans-activating activity of GAL4BD-APC2 fusion protein ("self-activation").
• FIGS. 5A - 5F illustrates the function of ROCl in yeast.
• FIG. 5A illustrates that ScROCl is an essential gene. Twenty tetrads from a +/rocl :kanR sporulated culture were dissected onto YPD plates, as shown.
• FIG. 5B illustrates depletion of ScROClp results in multi-budded cells.
• GAL-HA3-ScROCl haploids were cultured in 2% galactose plus 2% raffmose (top panels) or 2% glucose (bottom panels) for 24 hours. DNA was stained using Hoechst dye.
• FIG. 5C illustrates depletion of ScROClp.
• GAL-HA3 -ScROCl yeast cells were grown in either 2%, 0.05% galactose plus 2% raffmose or 2% glucose for different length of time as indicated. Cell lysates were resolved on an SDS- PAGE gel, transferred to nitrocellulose and blotted with anti-HA antibody to detect HA3-ROC1.
• FIG. 5D illustrates that ScROCl interacts with all yeast cullins.
• HF7c cells his3-200, leu2-3, trpl-901, GAL4-lacZ, GAL1-HIS3 were co-transformed with plasmids expressing indicated proteins (key). Protein-protein interactions were detennined by the yeast two-hybrid assay as described in FIG 1A.
• FIG. 5E shows that human ROCl and human ROC2 can rescue the multibudded phenotype resulting from ScROClp deletion.
• GAL-HA3- ScROCl haploids were transformed with pADH-414 vector, pADH-414-ScROCl, pADH- ScAPCl 1, pADH-hROCl or pADH-hROC2.
• Transformants were streaked onto selective plates containing 2% glucose and grown for 24 hours when the yeast cells demonstrate a multiple elongated phenotype. Cells were formaldehyde fixed before photography.
• FIG. 5F illustrates that Sic 1 p accumulates in yeast depleted of ScROClp.
• GAL-HA3-ScROCl/SICl-HA3 yeast cells were grown in either 0.05% galactose or 2% glucose for different length of time as indicated. Cell lysates were resolved on an SDS-PAGE gel, transferred to nitrocellulose and blotted with anti-HA antibody to detect Sicl-HA3 and with anti-actin antibody to detect action to verify equal protein loading.
• FIGS. 6A-6C illustrates that ROCl stimulates cullin-dependent ubiquitin ligase activity.
• FIG. 6 A illustrates that lysates (1 mg of total proteins) from human 293T cells transiently transfected with plasmids expressing indicated proteins were mixed with protein A beads linked to anti-HA antibodies. HA-immunocomplexes immobilized on the beads were washed and then mixed with purified El , E2 CD34 (unless otherwise idicated), 2 P-labeled ubiquitin (ub) and ATP.
• FIG. 6B illustrates that ubiquitin ligase activity was assayed as in (A) using lysates derived from cells transfected with plasmids expressing different combination of proteins as indicated.
• FIG. 6C illustrates in vivo ubiquitin ligase activity. Lysates from un-transfected human HeLa or 293T cells were immunoprecipated with antibodies to either ROCl, APC11 or CULl as indicated with (lane 4) or without competing peptide. Ubiquitin ligase activity was assayed as described herein.
• ROCl and ROC2 refer to the amino acid sequences of substantially purified ROCl and ROC2 obtained from any species, particularly mammalian, including bovine, ovine, porcine, murine, equine, and preferably human, from any source whether natural, synthetic, semi-synthetic, or recombinant.
• allelic sequence is an alternative fonn of the genes encoding ROCl and ROC2. Alleles may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
• protein herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
• the protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures.
• amino acid or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids. For example, homo- phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention.
• Amino acid also includes imino acid residues such as proline and hydroxyproline.
• the side chains may be in either the (R) or the (S) configuration. In the preferred embodiment, the amino acids are in the (S) or L- configuration.
• non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.
• Chemical blocking groups or other chemical substituents may also be added.
• Amino acid sequence refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragment thereof, and to naturally occurring or synthetic molecules. Fragments of ROCl and/or ROC2 are preferably about 5 to about 15 amino acids in length and retain the biological activity or the immunological activity of ROCl and/or ROC2.
• amino acid sequence is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, amino acid sequence, and like terms, are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
• PCR polymerase chain reaction
• the term "antibody” refers to intact molecules as well as fragments thereof, such as Fa, F(ab')2, and Fc, which are capable of binding the epitopic determinant.
• Antibodies that bind ROCl and/or ROC2 polypeptides can be prepared using intact polypeptides or fragments containing small peptides of interest as the immunizing antigen.
• the polypeptide or oligopeptide used to immunize an animal can be derived from the translation of RNA or synthesized chemically and can be conjugated to a carrier protein, if desired. Commonly used earners that are chemically coupled to peptides include bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin.
• the coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit).
• the term "antigenic determinant”, as used herein, refers to that fragment of a molecule (i.e., an epitope) that makes contact with a particular antibody.
• an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
• antisense refers to any composition containing nucleotide sequences which are complementary to a specific DNA or RNA sequence.
• antisense strand is used in reference to a nucleic acid strand that is complementary to the "sense” strand.
• Antisense molecules include peptide nucleic acids and may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to fo ⁇ n duplexes and block either transcription or translation. The designation “negative” is sometimes used in reference to the antisense strand, and “positive” is sometimes used in reference to the sense strand.
• complementarity refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing.
• sequence "A-G-T” binds to the complementary sequence "T-C-A.”
• Complementarity between two single-stranded molecules may be “partial”, in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between the single stranded molecules.
• the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
• a “deletion”, as used herein, refers to a change in the amino acid or nucleotide sequence and results in the absence of one or more amino acid residues or nucleotides.
• derivative refers to the chemical modification of a nucleic acid encoding or complementary to ROCl and/or ROC2 or the encoded ROCl and/or ROC2. Such modifications include, for example, replacement of hydrogen by an alkyl, acyl, or amino group.
• a nucleic acid derivative encodes a polypeptide which retains the biological or immunological function of the natural molecule.
• a derivative polypeptide is one which is modified by glycosylation, pegylation, or any similar process which retains the biological or immunological function of the polypeptide from which it was derived.
• homoology refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity).
• a partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to using the functional term "substantially homologous.”
• the inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization and the like) under conditions of low stringency.
• a substantially homologous sequence or hybridization probe will compete for and inhibit the binding of a completely homologous sequence to the target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction.
• the absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity). In the absence of non-specific binding, the probe will not hybridize to the second non-complementary target sequence.
• hybridization refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
• hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the fo ⁇ nation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be ftirther stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration.
• a hybridization complex may be fonned in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
• a solid support e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed.
• nucleic acid' or "oligonucleotide” or grammatical equivalents herein means at least two nucleotides covalently linked together.
• a nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage, et al, Tetraliedron, 49(10): 1925 (1993) and references therein; Letsinger, J. Org. Chem.. 35:3800 (1970); Sblul, et al, Eur. J. Biochem.. 81:579 (1977); Letsinger, et al, Nvicl. Acids Res..
• nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins, et al, Chem. Soc. Rev.. (1995) pp. 169-176).
• nucleic acid analogs are described in Rawls, Q & E News. June 2, 1997, page 35. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments.
• nucleic acids and analogs can be made.
• mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
• the nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence.
• the nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc.
• nucleic acid candidate bioactive agents may be naturally occumng nucleic acids, random nucleic acids, or "biased" random nucleic acids.
• digests of procaryotic or eukaryotic genomes may be used as is outlined above for proteins.
• Nucleic acid sequence refers to an oligonucleotide, nucleotide, or polynucleotide, and fragments thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.
• “Fragments” are those nucleic acid sequences which are greater than 60 nucleotides than in length, and most preferably includes fragments that are at least 100 nucleotides or at least 1000 nucleotides, and at least 10,000 nucleotides in length.
• oligonucleotide refers to a nucleic acid sequence of at least about 6 nucleotides to about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20 to 25 nucleotides, which can be used in PCR amplification or a hybridization assay, or a microarray.
• oligonucleotide is substantially equivalent to the terms “amplimers”, “primers”, “oligomers”, and “probes”, as commonly defined in the art.
• sample is used in its broadest sense.
• a biological sample suspected of containing nucleic acid encoding ROCl and/or ROC2, or fragments thereof, or ROCl and/or ROC2 itself may comprise a bodily fluid, extract from a cell, chromosome, organelle, or membrane isolated from a cell, a cell, genomic DNA, RNA, or cDNA (in solution or bound to a solid support, a tissue, a tissue print, and the like).
• stringent conditions or “stringency”, as used herein, refer to the conditions for hybridization as defined by the nucleic acid, salt, and temperature.
• conditions are well known in the ait and may be altered in order to identify or detect identical or related polynucleotide sequences.
• Numerous equivalent conditions comprising either low or high stringency depend on factors such as the length and nature of the sequence (DNA, RNA, base composition), nature of the target (DNA, RNA, base composition), milieu (in solution or immobilized on a solid substrate), concentration of salts and other components (e.g., formamide, dextran sulfate and/or polyethylene glycol), and temperature of the reactions (within a range from about 5° C. below the melting temperature of the probe to about 20° C. to 25° C. below the melting temperature).
• concentration of salts and other components e.g., formamide, dextran sulfate and/or polyethylene glycol
• temperature of the reactions within a range from about 5° C. below the melting temperature of the probe to about 20° C. to 25° C. below the melting temperature.
• One or more factors may be varied to generate conditions of either low
• substitution refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
• Transformation describes a process by which exogenous DNA enters and changes a recipient cell. It may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the type of host cell being transformed and may include, but is not limited to, viral infection, electroporation, heat shock, lipofection, and particle bombardment.
• Such "transformed” cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. They also include cells which transiently express the inserted DNA or RNA for limited periods of time.
• Polynucleotides of the present invention include those coding for proteins homologous to, and having essentially the same biological properties as, the proteins disclosed herein, and particularly the DNA disclosed herein as SEQ ID NO:l and encoding the protein ROCl given herein SEQ ID NO:2; as well as the DNA disclosed herein as SEQ ID NO: 3 and encoding the protein ROC2 given herein as SEQ ID NO:4.
• This definition is intended to encompass natural allelic sequences thereof.
• isolated DNA or cloned genes of the present invention can be of any species of origin, including mouse, rat, rabbit, cat, porcine, and human, but are preferably of mammalian origin.
• polynucleotides that hybridize to DNA disclosed herein as SEQ ID NO:l (or fragments or derivatives thereof which serve as hybridization probes as discussed below) and which code on expression for a protein of the present invention e.g., a protein according to SEQ ID NO:2
• polynucleotides that hybridize to DNA disclosed herein as SEQ ID NO:3 (or fragments or derivatives thereof which serve as hybridization probes as discussed below) and which code on expression for a protein of the present invention e.g., a protein according to SEQ ID NO:4
• Conditions which will permit other polynucleotides that code on expression for a protein of the present invention to hybridize to the DNA of SEQ ID NO:l or SEQ ID NO: 3 disclosed herein can be detem-iined in accordance with known techniques.
• hybridization of such sequences may be carried out under conditions of reduced stringency, medium stringency or even stringent conditions (e.g., conditions represented by a wash stringency of 35-40% Formamide with 5x Denhardt's solution, 0.5% SDS and lx SSPE at 37°C; conditions represented by a wash stringency of 40-45%> Formamide with 5x Denhardt's solution, 0.5% SDS, and lx SSPE at 42°C; and conditions represented by a wash stringency of 50%o Formamide with 5x Denhardt's solution, 0.5% SDS and lx SSPE at 42°C, respectively) to DNA of SEQ ID NO:l or SEQ ID NO: 3 disclosed herein in a standard hybridization assay.
• sequences which code for proteins of the present invention and which hybridize to the DNA of SEQ ID NO:l or SEQ ID NO: 3 disclosed herein will be at least 75% homologous, 85% homologous, and even 95% homologous or more with SEQ ID NO:l or SEQ ID NO:3, respectively.
• polynucleotides that code for proteins of the present invention or polynucleotides that hybridize to that as SEQ ID NO:l or SEQ ID NO:3, but which differ in codon sequence from SEQ ID NO:l or SEQ ID NO:3 due to the degeneracy of the genetic code, are also an aspect of this invention.
• the degeneracy of the genetic code which allows different nucleic acid sequences to code for the same protein or peptide, is well known in the literature. See, e.g., U.S. Patent No. 4,757,006 to Toole et al. at Col. 2, Table 1.
• nucleotide sequences which encode ROCl and/or ROC2 and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occumng ROCl and/or ROC2 under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding ROCl and/or ROC2 or its derivatives possessing a substantially different codon usage. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
• RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
• ROC nucleic acids defined as polynucleotides encoding ROC proteins of fragments thereof), or ROC proteins (as defined above) are initially identified by substantial nucleic acid and/or amino acid sequence identity or similarity to the sequence(s) provided herein.
• ROC nucleic acids or ROC proteins have sequence identity or similarity to the sequences provided herein as described below and one or more of the ROC protein bioactivities as further described herein.
• sequence identity or similarity can be based upon the overall nucleic acid or amino acid sequence.
• a number of different programs can be used to identify whether a protein (or nucleic acid as discussed below) has sequence identity or similarity to a known sequence.
• Sequence identity and/or similarity is determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2, 482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48,443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85,2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the Best Fit sequence program described by Devereux et al, Nucl Acid Res.
• percent identity is calculated by FastDB based upon the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30, "Current Methods in Sequence Comparison and -Analysis," Macromolecule Sequencing and Synthesis. Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.
• PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol 35, 351-360 (1987); the method is similar to that described by Higgins & Sharp CABIOS 5, 151-153 (1989).
• Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.
• BLAST Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993).
• a particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al., Methods in Enzymology. 266, 460-480 (1996); http://blast.wustl/edu/blast/ README.html.
• WU-BLAST-2 uses several search parameters, most of which are set to the default values.
• the HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
• Gapped BLAST uses BLOSUM-62 substitution scores; threshold T parameter set to 9; the two-hit method to trigger ungapped extensions; charges gap lengths of k a cost of 10+/c; X u set to 16, and X % set to 40 for database search stage and to 67 for the output stage of the algorithms. Gapped alignments are triggered by a score corresponding to ⁇ 22 bits. A percentage amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region.
• the "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU- Blast-2 to maximize the alignment score are ignored).
• percent (%) nucleic acid sequence identity with respect to the coding sequence of the polypeptides identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the coding sequence of the cell cycle protein.
• a preferred method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.
• the alignment may include the introduction of gaps in the sequences to be aligned.
• sequences which contain either more or fewer amino acids than the protein encoded by the sequences in the Figures it is understood that in one embodiment, the percentage of sequence identity will be detemiined based on the number of identical amino acids in relation to the total number of amino acids. Thus, for example, sequence identity of sequences shorter than that shown in the Figure, as discussed below, will be dete ⁇ nined using the number of amino acids in the shorter sequence, in one embodiment. In percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as, insertions, deletions, substitutions, etc.
• identities are scored positively (+1) and all forms of sequence variation including gaps are assigned a value of "0", which obviates the need for a weighted scale or parameters as described below for sequence similarity calculations.
• Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100. The "longer" sequence is the one having the most actual residues in the aligned region.
• the invention also encompasses production of DNA sequences, or fragments thereof, which encode ROCl and/or ROC2 and its derivatives, entirely by synthetic chemistry.
• the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents that are well known in the art.
• synthetic chemistry may be used to introduce mutations into a sequence encoding ROCl and/or ROC2 or any fragment thereof.
• nucleotide sequence as disclosed herein in SEQ ID NO:l or SEQ ID NO:3 can be used to generate hybridization probes which specifically bind to the DNA of the present invention or to mRNA to determine the presence of amplification or overexpression of the proteins of the present invention.
• Methods for DNA sequencing which are well known and generally available in the art may be used to practice any of the embodiments of the invention.
• the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE® (US Biochemical Corp, Cleveland, Ohio), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,
• the process is automated with machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, Mass.) and the -ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
• machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, Mass.) and the -ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
• the nucleic acid sequences encoding ROCl and/or ROC2 may be extended utilizing a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements.
• one method which may be employed, "restriction-site" PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, G. (1993) PCR Methods Applic. 2, 318-322).
• genomic DNA is first amplified in the presence of primer to a linker sequence and a primer specific to the known region.
• the amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one.
• Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
• a vector is a replicable DNA const ct. Vectors are used herein either to amplify DNA encoding the proteins of the present invention or to express the proteins of the present invention.
• -An expression vector is a replicable DNA construct in which a DNA sequence encoding the proteins of the present invention is operably linked to suitable control sequences capable of effecting the expression of proteins of the present invention in a suitable host. The need for such control sequences will vary depending upon the host selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. Amplification vectors do not require expression control domains. All that is needed is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants.
• Vectors comprise plasmids, viruses (e.g., adenovims, cytomegalovirus), phage, retroviruses and integratable DNA fragments (i.e., fragments integratable into the host genome by recombination).
• the vector replicates and functions independently of the host genome, or may, in some instances, integrate into the genome itself.
• Expression vectors should contain a promoter and RNA binding sites which are operably linked to the gene to be expressed and are operable in the host organism. DNA regions are operably linked or operably associated when they are functionally related to each other.
• a promoter is operably linked to a coding sequence if it controls the transcription of the sequence;
• a ribosome binding site is operably linked to a coding sequence if it is positioned so as to pe ⁇ nit translation.
• operably linked means contiguous and, in the case of leader sequences, contiguous and in reading phase.
• Transformed host cells are cells which have been transfo ⁇ ned or transfected with vectors containing DNA coding for proteins of the present invention need not express protein.
• Suitable host cells include prokaryotes, yeast cells, or higher eukaryotic organism cells.
• Prokaiyote host cells include gram negative or gram positive organisms, for example Escherichia coli (E. coli) or Bacilli.
• Higher eukaryotic cells include established cell lines of mammalian origin as described below.
• Exemplary host cells are E. coli W3110 (ATCC 27,325), E. coli B, E. coli X1776 (ATCC 31,537), E. coli 294 (ATCC 31,446).
• a broad variety of suitable prokaryotic and microbial vectors are available.
• E. coli is typically transformed using pBR322. See Bolivar et al, Gene 2, 95 (1977).
• Promoters most commonly used in recombinant microbial expression vectors include the beta-lactamase (penicillinase) and lactose promoter systems (Chang et al, Nature.275, 615 (1978); and Goeddel et al, Nature 281, 544 (1979), a tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980) and ⁇ PO App. Publ. No. 36,776) and the tac promoter (H. De Boer et al, Proc. Natl. Acad. Sci. USA 80, 21 (1983).
• the promoter and Shine-Dalgarno sequence are operably linked to the DNA of the present invention, i.e., they are positioned so as to promote transcription of the messenger RNA from the DNA.
• Expression vectors should contain a promoter which is recognized by the host organism. This generally means a promoter obtained from the intended host.
• Promoters most commonly used in recombinant microbial expression vectors include the beta-lactamase (penicillinase) and lactose promoter systems (Chang et al, Nature 275, 615 (1978); and Goeddel et al, Nature 281, 544 (1979), a tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980) and EPO App. Publ. No. 36,776) and the tac promoter (H. De Boer et al., Proc. Natl. Acad. Sci. USA 80, 21 (1983). While these are commonly used, other microbial promoters are suitable.
• nucleotide sequences of many have been published, enabling a skilled worker to operably ligate them to DNA encoding the protein in plasmid or viral vectors (Siebenlist et al., Cell 20, 269 (1980).
• the promoter and Shine-Dalgarno sequence are operably linked to the DNA encoding the desired protein, i.e., they are positioned so as to promote transcription of the protein messenger RNA from the DNA.
• Eukaryotic microbes such as yeast cultures may be transformed with suitable protein-encoding vectors. See e.g., U.S. Patent No. 4,745,057. Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms, although a number of other strains are commonly available.
• Yeast vectors may contain an origin of replication from the 2 micron yeast plasmid or anautonomously replicating sequence (ARS), a promoter, DNA encoding the desired protein, sequences for polyadenylation and transcription te ⁇ nination, and a selection gene.
• ARS autonomously replicating sequence
• An exemplary plasmid is YRp7, (Stinchcomb et al., Nature 282, 39 (1979); Kingsman et al., Gene 1, 141 (1979); Tschemper et al., Gene 10, 157 (1980).
• This plasmid contains the t ⁇ l gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85, 12 (1977). The presence of the t l lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
• Suitable promoting sequences in yeast vectors include the promoters for metallothionein, 3- ⁇ hospho-glycerate kinase (Hitzeman et al, J. Biol. Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al, J. Adv. Enzyme Reg.
• Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream from the gene to be expressed, along with a ribosome binding site, RNA splice site (if intron-containing genomic DNA is used), a polyadenylation site, and a transcriptional termination sequence.
• the transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells are often provided by viral sources.
• promoters are derived from polyoma, Adenovims 2, and Simian Vims 40 (SV40). See, e.g., U.S. Patent No. 4,599,308.
• the early and late promoters are useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication. See Fiers et al., Nature 273, 113 (1978).
• the protein promoter, control and/or signal sequences may also be used, provided such control sequences are compatible with the host cell chosen.
• An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral source (e.g. Polyoma, Adenovims, VSV, or BPV), or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient.
• Host cells such as insect cells (e.g., cultured Spodoptera frugiperda cells) and expression vectors such as the baculorivus expression vector (e.g., vectors derived from Autographa califo nica MNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV) may be employed to make proteins useful in carrying out the present invention, as described in U.S. Patents Nos. 4,745,051 and 4,879,236 to Smith et al.
• insect cells e.g., cultured Spodoptera frugiperda cells
• expression vectors such as the baculorivus expression vector
• the baculorivus expression vector e.g., vectors derived from Autographa califo nica MNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV
• a baculovims expression vector comprises a baculovirus genome containing the gene to be expressed inserted into the polyhedrin gene at a position ranging from the polyhedrin transcriptional start signal to the ATG start site and under the transcriptional control of a baculovims polyhedrin promoter.
• a number of viral-based expression systems may be utilized, hi cases where an adenovims is used as an expression vector, sequences encoding ROCl and or ROC2 may be ligated into an adenovims transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing ROCl and/or ROC2 in infected host cells (Logan, J. and Shenlc, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659).
• transcription enhancers such as the Rous sarcoma vims (RSV) enhancer, may be used to increase expression in mammalian host cells.
• RSV Rous sarcoma vims
• a selectable marker is dihydrofolate reductase (DHFR) or thymidine kinase.
• DHFR dihydrofolate reductase
• thymidine kinase thymidine kinase.
• peptides containing such deletions or substitutions are a further aspect of the present invention.
• one or more amino acids of a peptide sequence may be replaced by one or more other amino acids wherein such replacement does not affect the function of that sequence.
• Such changes can be guided by known similarities between amino acids in physical features such as charge density, hydrophobicity/hydrophilicity, size and configuration, so that amino acids are substituted with other amino acids having essentially the same functional properties.
• Ala may be replaced with Val or Ser; Val may be replaced with Ala, Leu, Met, or lie, preferably Ala or Leu; Leu may be replaced with Ala, Val or He, preferably Val or lie; Gly may be replaced with Pro or Cys, preferably Pro; Pro may be replaced with Gly, Cys, Ser, or Met, preferably Gly, Cys, or Ser; Cys may be replaced with Gly, Pro, Ser, or Met, preferably Pro or Met; Met may be replaced with Pro or Cys, preferably Cys; His may be replaced with Phe or Gin, preferably Phe; Phe may be replaced with His, Tyr, or T ⁇ , preferably His or Tyr; Tyr may be replaced with His, Phe or T ⁇ , preferably Phe or T ; T ⁇ may be replaced with Phe or Tyr, preferably Tyr; Asn may be replaced with Gin or Ser, preferably Gin; KGln may be replaced with His, Lys, Glu, Asn, or Ser, preferably Asn or Ser; Ser may be replaced with Gin,
• the present invention provides isolated and purified ROCl and ROC2 proteins, such as mammalian (or more preferably human) ROCl and ROC2.
• ROCl and ROC2 proteins such as mammalian (or more preferably human) ROCl and ROC2.
• Such proteins can be purified from host cells which express the same, in accordance with known techniques, or even manufactured synthetically.
• Nucleic acids of the present invention constructs containing the same and host cells that express the encoded proteins are useful for making proteins of the present invention.
• Proteins of the present invention are useful as immunogens for making antibodies as described herein, and these antibodies and proteins provide a "specific binding pair.” Such specific binding pairs are useful as components of a variety of immunoassays and purification techniques, as is known in the art.
• the proteins of the present invention are of known amino acid sequence as disclosed herein, and hence are useful as molecular weight markers in determining the molecular weights of proteins of unknown structure.
• Specific initiation signals may also be used to achieve more efficient translation of sequences encoding ROCl and or ROC2. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding ROCl and/or ROC2, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).
• a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
• modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
• Post- translational processing which cleaves a "prepro" form of the protein may also be used to facilitate conect insertion, folding and/or function.
• Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure the correct modification and processing of the foreign protein.
• ATCC American Type Culture Collection
• cell lines which stably express ROCl and/or ROC2 may be transformed using expression vectors which may contain viral origins of replication and or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The pmpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type. Any number of selection systems may be used to recover transfomied cell lines.
• heipes simplex vims thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) genes which can be employed in tk- or aprt- cells, respectively.
• antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
• npt which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Muny, supra). Additional selectable genes have been described, for example, t ⁇ B, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci.
• transfomied cells containing sequences encoding ROCl and or ROC2 can be identified by the absence of marker gene function.
• a marker gene can be placed in tandem with a sequence encoding ROCl and/or ROC2 under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
• ROCl and/or ROC2 and express ROCl and or ROC2 may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and or quantification of nucleic acid or protein.
• the presence of polynucleotide sequences encoding ROCl and/orROC2 can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding ROCl and/or ROC2.
• Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequences encoding ROCl and/or ROC2 to detect transformants containing DNA or RNA encoding ROCl and/or ROC2.
• ROCl and/or ROC2 A variety of protocols for detecting and measuring the expression of ROCl and/or ROC2, using either polyclonal or monoclonal antibodies specific for the protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
• ELISA enzyme-linked immunosorbent assay
• RIA radioimmunoassay
• FACS fluorescence activated cell sorting
• a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non- interfering epitopes on ROCl and/or ROC2 is prefened, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul, Minn.) and Maddox, D. E. et al. (1983; J.
• Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding ROCl and or ROC2 include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide.
• the sequences encoding ROCl and/or ROC2, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
• RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
• T7, T3, or SP6 RNA polymerase
• Suitable reporter molecules or labels include radionuchdes, enzymes, fluorescent, chemiluminescent, or cliromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
• Host cells transformed with nucleotide sequences encoding ROCl and/or ROC2 may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
• the protein produced by a transfomied cell may be secreted or contained intracellularly depending on the sequence and/or the vector used.
• expression vectors containing polynucleotides which encode ROCl and/or ROC2 may be designed to contain signal sequences which direct secretion of ROCl and or ROC2 through a prokaryotic or eukaryotic cell membrane.
• constmctions may be used to join sequences encoding ROCl and/or ROC2 to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins.
• purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immuno globulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Co ⁇ ., Seattle, Wash.).
• cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, Calif.) between the purification domain and ROCl and or ROC2 may be used to facilitate purification.
• One such expression vector provides for expression of a fusion protein containing ROCl and/or ROC2 and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMAC (immobilized metal ion affinity cl romatography) as described in Porath, J. et al. (1992, Prot. Exp. Purif.
• IMAC immobilized metal ion affinity cl romatography
• enterokinase cleavage site provides a means for purifying ROCl and/or ROC2 from the fusion protein.
• fragments of ROCl and/or ROC2 may be produced by direct peptide synthesis using solid-phase techniques (Merrifield J. (1963) J. Am. Chem. Soc. 85, 2149-2154). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Various fragments of ROCl and or ROC2 may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.
• Antibodies that specifically bind to the proteins of the present invention are useful for a variety of diagnostic pu ⁇ oses.
• Antibodies to ROCl and/or ROC2 may be generated using methods that are well known in the ait. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies, (i.e., those which inhibit dimer fonnation) are especially prefened for therapeutic use.
• various hosts including goats, rabbits, rats, mice, humans, and others, may be immunized by injection with ROCl and/or ROC2 or any fragment or oligopeptide thereof which has immunogenic properties.
• various adjuvants may be used to increase immunological response.
• adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
• BCG Bacilli Calmette-Guerin
• Coiynebacterium parvum are especially preferable.
• the oligopeptides, peptides, or fragments used to induce antibodies to ROCl and/or ROC2 have an amino acid sequence consisting of at least five amino acids and more preferably at least 10 amino acids. It is also preferable that they are identical to a portion of the amino acid sequence of the natural protein, and they may contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of ROCl and/or ROC2 amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule.
• Monoclonal antibodies to ROCl and/or ROC2 may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. See, e.g., Kohler, G. et al. (1975) Nature, 256, 495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81, 31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. USA 80, 2026-2030; Cole, S. P. et al. (1984) Mol.
• Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci, 86, 3833-3837; Winter, G. et al. (1991) Nature 349,:293-299.
• Antibody fragments which contain specific binding sites for ROCl and/or ROC2 may also be generated.
• such fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
• Fab expression libraries may be constmcted to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. See Huse, W. D. et al. (1989) Science 254,1275-1281.
• Various immunoassays may be used for screening to identify antibodies having the desired specificity.
• Such immunoassays typically involve the measurement of complex formation between ROCl and/or ROC2 and its specific antibody.
• a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering ROCl and/or ROC2 epitopes is prefened, but a competitive binding assay may also be employed (Maddox, supra).
• Antibodies may be conjugated to a solid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as precipitation.
• Antibodies may likewise be conjugated to detectable groups such as radiolabels (e.g., 5 S, 125 I, 131 I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein) in accordance with known techniques.
• radiolabels e.g., 5 S, 125 I, 131 I
• enzyme labels e.g., horseradish peroxidase, alkaline phosphatase
• fluorescent labels e.g., fluorescein
• Kits for determining if a sample contains proteins of the present invention will include at least one reagent specific for detecting the presence or absence of the protein. Diagnostic kits for carrying out antibody assays may be produced in a number of ways.
• the diagnostic kit comprises (a) an antibody which binds proteins of the present invention conjugated to a solid support and (b) a second antibody which binds proteins of the present invention conjugated to a detectable group.
• the reagents may also include ancillary agents such as buffering agents and protein stabilizing agents, e.g., polysaccharides and the like.
• the diagnostic kit may further include, where necerney, other members of the signal- producing system of which system the detectable group is a member (e.g., enzyme substrates), agents for reducing background interference in a test, control reagents, apparams for conducting a test, and the like.
• a second embodiment of a test kit comprises (a) an antibody as above, and (b) a specific binding partner for the antibody conjugated to a detectable group. Ancillary agents as described above may likewise be included.
• the test kit may be packaged in any suitable manner, typically with all elements in a single container along with a sheet of printed instmctions for canying out the test.
• Assays for detecting the polynucleotides encoding ROCl or ROC2 in a cell, or the extent of amplification thereof typically involve, first, contacting the cells or extracts of the cells containing nucleic acids therefrom with an oligonucleotide that specifically binds to ROCl or ROC2 polynucleotide as given herein (typically under conditions that permit access of the oligonucleotide to intracellular material), and then detecting the presence or absence of binding of the oligonucleotide thereto.
• any suitable assay format may be employed (--see, e.g., U.S. Patent No. 4,358,535 to Falkow et al.; U.S. Patent No.
• Antisense oligonucleotides and nucleic acids that express the same may be made in accordance with conventional techniques. See, e.g., U.S. Patent No. 5.023,243 to Tullis; U.S. Patent No. 5,149,797 to Pederson et al.
• the length of the antisense oligonucleotide i.e., the number of nucleotides therein
• the antisense oligonucleotide will be from 8, 10 or 12 nucleotides in length up to 20, 30, or 50 nucleotides in length.
• Such antisense oligonucleotides may be oligonucleotides wherein at least one, or all, or the intemucleotide bridging phosphate residues are modified phosphates, such as methyl phosphonates, methyl phosphonothioates, phosphoromo ⁇ holidates, phosphoropiperazidates and phosphoramidates. For example, every other one of the intemucleotide bridging phosphate residues may be modified as described.
• such antisense oligonucleotides are oligonucleotides wherein at least one, or all, of the nucleotides contain a 2' loweralkyl moiety (e.g., C ⁇ -C 4 , linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl).
• every other one of the nucleotides may be modified as described. See also P. Furdon et al., Nucleic Acids Res. 17, 9193- 9204 (1989); S. Agrawal et al, Proc. Natl Acad.
• the ROC proteins, nucleic acids, variants, modified proteins, cells and/or transgenics containing the ROC nucleic acids or proteins are used in screening assays. Identification of the ROC proteins provided herein permits the design of dmg screening assays for compounds that bind or interfere with the binding to the ROC proteins and for compounds which modulate ROC activity.
• the assays described herein preferably utilize the human ROC proteins, although other mammalian proteins may also be used, including rodents (mice, rats, hamsters, guinea pigs, etc.), farm animals (cows, sheep, pigs, horses, etc.) and primates. These latter embodiments may be prefened in the development of animal models of human disease.
• rodents mice, rats, hamsters, guinea pigs, etc.
• farm animals cows, sheep, pigs, horses, etc.
• primates primates.
• variant or derivative ROC proteins may be used, including deletion ROC proteins as outlined above.
• the methods comprise combining a ROC protein and a candidate bioactive agent, and determining the binding of the candidate agent to the ROC proteins. In other embodiments, further discussed below, binding interference or bioactivity is determined.
• candidate bioactive agent or "exogeneous compound” as used herein describes any molecule, e.g., protein, small organic molecule, carbohydrates (including polysaccharides), polynucleotide, lipids, etc.
• assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations.
• one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.
• positive controls i.e. the use of agents known to alter ROC activity, may be used.
• Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons.
• Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
• the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
• Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
• Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.
• a library of different candidate bioactive agents are used.
• the library should provide a sufficiently structurally diverse population of randomized agents to effect a probabilistically sufficient range of diversity to allow binding to a particular target.
• an interaction library should be large enough so that at least one of its members will have a structure that gives it affinity for the target.
• a diversity of 10 -10 different antibodies provides at least one combination with sufficient affinity to interact with -most potential antigens faced by an organism. Published in vitro selection techniques have also shown that a library size of 10 7 to 10 s is sufficient to find stmctures with affinity for the target.
• a library of all combinations of a peptide 7 to 20 amino acids in length has the potential to code for 20 7 (10 9 ) to 20 20 .
• the present methods allow a "working" subset of a theoretically complete interaction library for 7 amino acids, and a subset of shapes for the 20 20 library.
• at least 10 6 preferably at least 10 7 , more preferably at least 10 8 and most preferably at least 10 9 different sequences are simultaneously analyzed in the subject methods. Prefened methods maximize library size and diversity.
• the candidate bioactive agents are proteins.
• the candidate bioactive agents are naturally occurring proteins or fragments of naturally occurring proteins.
• cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts may be used.
• libraries of prokaryotic and eukaryotic proteins may be made for screening in the systems described herein.
• Particularly prefened in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being prefened, and human proteins being especially prefened.
• the candidate bioactive agents are peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being prefened, and from about 7 to about 15 being particulaiiy prefened.
• the peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or "biased” random peptides.
• randomized or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may inco ⁇ orate any nucleotide or amino acid at any position.
• the synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the fom ation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.
• the library is fully randomized, with no sequence preferences or constants at any position.
• the library is biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities.
• the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.
• the candidate bioactive agents are nucleic acids.
• the candidate bioactive agents are organic chemical moieties, a wide variety of which are available in the literature.
• portions of ROC proteins are utilized; in a prefened embodiment, portions having ROC activity are used.
• ROC activity is as described herein and includes binding activity to cullins as outlined herein.
• the assays described herein may utilize either isolated ROC proteins or cells comprising the ROC proteins.
• the ROC proteins or the candidate agent is non-diffusibly bound to an insoluble support having isolated sample receiving areas (e.g. a microtiter plate, an anay, etc.).
• the insoluble supports may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening.
• the surface of such supports may be solid or porous and of any convenient shape.
• suitable insoluble supports include microtiter plates, anays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose, TEFLON ® , etc.
• Microtiter plates and anays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. In some cases magnetic beads and the like are included.
• the particular manner of binding of the composition is not cmcial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable.
• Prefened methods of binding include the use of antibodies (which do not sterically block important sites on the protein when the protein is bound to the support), direct binding to "sticky" or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or agent, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine semm albumin (BSA), casein or other innocuous protein or other moiety.
• BSA bovine semm albumin
• screening assays wherein solid supports are not used; examples of such are described below.
• the ROC proteins is bound to the support, and a candidate bioactive agent is added to the assay.
• the candidate agent is bound to the support and the ROC proteins is added.
• Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc. Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays may be used for this pu ⁇ ose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays, and the like.
• the determination of the binding of the candidate bioactive agent to the ROC proteins may be done in a number of ways.
• the candidate bioactive agent is labelled, and binding determined directly. For example, this may be done by attaching all or a portion of the ROC proteins to a solid support, adding a labelled candidate agent (for example a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support.
• a labelled candidate agent for example a fluorescent label
• washing off excess reagent for example a fluorescent label
• determining whether the label is present on the solid support.
• Various blocking and washing steps may be utilized as is known in the art.
• labeled herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g. radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc.
• Specific binding molecules include pairs, such as biotin and streptavidm, digoxin and antidigoxin etc.
• the complementary member would normally be labeled with a molecule which provides for detection, in accordance with -known procedures.
• the label can directly or indirectly provide a detectable signal, h some embodiments, only one of the components is labeled.
• the proteins may be labeled at tyrosine positions using 125 I, or with fluorophores.
• more than one component may be labeled with different labels; using 125 I for the proteins, for example, and a fluorophor for the candidate agents.
• the binding of the candidate bioactive agent is detennined through the use of competitive binding assays.
• the competitor is a binding moiety known to bind to the target molecule (i.e. ROC proteins), such as an antibody, peptide, binding partner, ligand, etc.
• the competitor is a cullin.
• This assay can.be used to determine candidate agents which interfere with binding between ROC proteins and its biological binding partners.
• Interference of binding as used herein means that native binding of the ROC proteins differs in the presence of the candidate agent. The binding can be eliminated or can be with a reduced affinity. Therefore, in one embodiment, interference is caused by, for example, a conformation change, rather than direct competition for the native binding site.
• the candidate bioactive agent is labeled.
• Either the candidate bioactive agent, or the competitor, or both, is added first to the protein for a time sufficient to allow binding, if present.
• Incubations may be performed at any temperature which facilitates optimal activity, typically between 4 and 40 C . Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.
• the competitor is added first, followed by the candidate bioactive agent.
• Displacement of the competitor is an indication that the candidate bioactive agent is binding to the ROC proteins and thus is capable of binding to, and potentially modulating, the activity of the ROC proteins.
• either component can be labeled.
• the candidate bioactive agent is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor may indicate that the bioactive agent is bound to the ROC proteins with a higher affinity.
• the methods comprise differential screening to identity bioactive agents that are capable of modulating the activity of the ROC proteins. Such assays can be done with the ROC proteins or cells comprising said ROC proteins.
• the methods comprise combining an ROC proteins and a competitor in a first sample.
• a second sample comprises a candidate bioactive agent, an ROC proteins and a competitor.
• the binding of the competitor is detennined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the ROC proteins and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the ROC proteins.
• a prefened embodiment utilizes differential screening to identify dmg candidates that bind to the native ROC proteins, but cannot bind to modified ROC proteins.
• the structure of the ROC proteins may be modeled, and used in rational d g design to synthesize agents that interact with that site.
• Dmg candidates that affect cell cycle bioactivity are also identified by screening drugs for the ability to either enhance or reduce the activity of the protein.
• Positive controls and negative controls may be used in the-assays.
• Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein. Following incubation, all samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to dete ⁇ nine the amount of bound compound.
• reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding.
• methods for screening for a bioactive agent capable of modulating the activity of ROC proteins comprise the steps of adding a candidate bioactive agent to a sample of a ROC proteins (or cells comprising a ROC proteins) and determining an alteration in the biological activity of the ROC proteins.
• “Modulating the activity of a ROC proteins” includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present.
• the candidate agent should both bind to the ROC protein (although this may not be necessary), and alter its biological or biochemical activity as defined herein.
• the methods include both in vitro screening methods, as are generally outlined above, and in vivo screening of cells for alterations in the presence, distribution, activity or amount of ROC proteins.
• the methods comprise combining a ROC protein and a candidate bioactive agent, and evaluating the effect on the bioactivity of the ROC proteins.
• ROC protein activity or grammatical equivalents herein is meant at least one of the ROC proteins' biological activities, including, but not limited to, the proteins' ability to bind cullins (including, but not limited to, cullin 1, 2, 3, 4A and 5), its activity in ligating ubiquitin and the ubiquitin-dependent proteolytic process, its role in SICp degradation, and any other activity of ROC proteins as described herein, etc.
• the activity of the ROC proteins is decreased; in another prefened embodiment, the activity of the ROC proteins is increased.
• bioactive agents that are antagonists are prefened in some embodiments, and bioactive agents that are agonists may be prefened in other embodiments.
• the invention provides methods for screening for bioactive agents capable of modulating the activity of an ROC proteins.
• the methods comprise adding a candidate bioactive agent, as defined above, to a cell comprising ROC proteins.
• Prefened cell types include almost any cell.
• the cells contain a recombinant nucleic acid that encodes a ROC protein.
• a library of candidate agents are tested on a plurality of cells.
• Detection of ROC activity may be done as will be appreciated by those in the art. There are a number of parameters that may be evaluated or assayed to allow the detection of alterations in ROC bioactivity.
• the measurements can be determined wherein all of the conditions are the same for each measurement, or under various conditions, with or without bioactive agents, etc.
• measurements of ROC activity can be determined in a cell or cell population wherein a candidate bioactive agent is present and wherein the candidate bioactive agent is absent.
• the measurements of ROC activity are determined wherein the condition or environment of the cell or populations of cells differ from one another.
• the cells may be evaluated in the presence or absence or previous or subsequent exposure of physiological signals, for example hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e. cell-cell contacts).
• apopulation of cells or “library of cells” herein is meant at least two cells, with at least about 10 3 being prefened, at least about 10 ⁇ being particularly prefened, and at least about 10 s to 10 9 being especially prefened.
• the population or sample can contain a mixture of different cell types from either primary or secondary cultures although samples containing only a single cell type are prefened, for example, the sample can be from a cell line, particularly tumor cell lines.
• cells that are replicating or proliferating are used; this may allow the use of refroviral vectors for the introduction of candidate bioactive agents.
• non-replicating cells may be used, and other vectors (such as adenovims and lentivims vectors) can be used.
• the cells are compatible with dyes and antibodies.
• Prefened cell types for use in the invention include, but are not limited to, mammalian cells, including animal (rodents, including mice, rats, hamsters and gerbils), primates, and human cells, particularly including tumor cells of all types, including breast, skin, lung, cervix, colonrectal, leukemia, brain, etc.
• mammalian cells including animal (rodents, including mice, rats, hamsters and gerbils), primates, and human cells, particularly including tumor cells of all types, including breast, skin, lung, cervix, colonrectal, leukemia, brain, etc.
• the proteins and nucleic acids provided herein can also be used for screening pu ⁇ oses wherein the protein-protein interactions of the ROC proteins can be identified.
• Genetic systems have been described to detect protein-protein interactions. The first work was done in yeast systems, namely the "yeast two- hybrid" system. The basic system requires a protein-protein interaction in order to turn on transcription of a reporter gene. Subsequent work was done in mammalian cells. See Fields et al., Nature 340, 245 (1989); Vasavada et al, Proc. Natl. Acad. Sci. USA 88, 10686 (1991); Fearon et al., Proc. Natl. Acad. Sci. USA 89, 7958 (1992); Dang et al, Mol.
• two nucleic acids are transfomied into a cell, where one is a "bait” such as the gene encoding a ROC proteins or a portion thereof, and the other encodes a test candidate. Only if the two expression products bind to one another will an indicator, such as a fluorescent protein, be expressed. Expression of the indicator indicates when a test candidate binds to the ROC proteins.
• an indicator such as a fluorescent protein
• the reverse can be performed. Namely, the ROC proteins provided herein can be used to identify new baits, or agents which interact with ROC proteins. Additionally, the two-hybrid system can 4o
• test candidate is added in addition to the bait and the ROC proteins encoding nucleic acids to determine agents which interfere with the bait, such as cullins.
• Bioactive agents i.e., compounds
• pharmacological activity are those compounds that are able to enhance or interfere with the activity of at least one of the ROC proteins.
• the compounds having the desired pharmacological activity may be administered in a phaniiaceutically acceptable carrier (i.e., a pha ⁇ naceutical formulation) to a host or subject Suitable subjects are preferably human subjects, but may also be other mammalian subjects, such as dogs, cats and livestock (i.e., for veterinary pu ⁇ oses).
• compositions of the present invention comprise compounds with pharmacological activity (as identified using methods of the present invention) in a pha ⁇ naceutically acceptable carrier.
• suitable pharmaceutical formulations include those suitable for inhalation, oral, rectal, topical, (including buccal, sublingual, dermal, vaginal and intraocular), parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular) and transdermal administration.
• the compositions may conveniently be presented in unit dosage fonn and may be prepared by any of the methods well known in the art. The most suitable route of administration in any given case may depend upon the anatomic location of the condition being treated in the subject, the nature and severity of the condition being treated, and the particular pharmacologically active compound which is being used.
• the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art.
• phannacologically active compounds or the physiologically acceptable salts thereof are typically admixed with, inter alia, an acceptable carrier.
• the carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the fonnulation and must not be deleterious to the patient.
• the canier may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose fonnulation, for example, a tablet, which may contain from 0.5%o to 99%o by weight of the active compound.
• One or more active compounds may be inco ⁇ orated in the formulations of the invention , which formulations may be prepared by any of the well known techniques of pha ⁇ nacy consisting essentially of admixing the components, optionally including one or more accessory therapeutic ingredients.
• Formulations suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion.
• Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association the active compound and a suitable carrier (which may contain one or more accessory ingredients as noted above).
• the fonnulations of the invention are prepared by uniformly and intimately admixing the active compound with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture.
• a tablet may be prepared by compressing or molding a powder or granules containing the active compound, optionally with one or more accessory ingredients.
• Compressed tablets may be prepared by compressing, in a suitable machine, the compound in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Molded tablets may be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder.
• Formulations for oral administration may optionally include enteric coatings known in the art to prevent degradation of the formulation in the stomach and provide release of the drug in the small intestine.
• Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the active compound in a flavored base, usually sucrose and acacia or fragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin or sucrose and acacia.
• Formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the active compound, which preparations are preferably isotonic with the blood of the intended recipient. These preparations may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient.
• Aqueous and non-aqueous sterile suspensions may include suspending agents and thickening agents.
• the formulations may be presented in unit ⁇ dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water- for-injection immediately prior to use.
• Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
• an injectable, stable, sterile composition comprising a compound of Formula (I), or a salt thereof, in a unit dosage fonn in a sealed container.
• the compound or salt is provided in the fonn of a lyophilizate which is capable of being reconstituted with a suitable pha ⁇ naceutically acceptable carrier to fonn a liquid composition suitable for injection thereof into a subject.
• the unit dosage form typically comprises from about 10 mg to about 10 grams of the compound or salt.
• emulsifying agent which is physiologically acceptable may be employed in sufficient quantity to emulsify the compound or salt in an aqueous carrier.
• emulsifying agent is phosphatidyl choline.
• Fonnulations suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.
• Formulations suitable for topical application to the skin preferably take the fomi of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil.
• Carriers which may be used include vaseline, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.
• Formulations suitable for transde ⁇ nal administration may be presented as discrete patches adapted to remain in intimate contact with the epidemiis of the recipient for a prolonged period of time.
• Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, e.g., Pharmaceutical Research 3, 318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound.
• the present invention provides liposomal formulations of the compounds disclosed herein and salts thereof.
• the technology for forming liposomal suspensions is well known in the art.
• the compound or salt thereof is an aqueous-soluble salt, using conventional liposome technology, the same may be incorporated into lipid vesicles. In such an instance, due to the water solubility of the compound or salt, the compound or salt will be substantially entrained within the hydrophilic center or core of the liposomes.
• the lipid layer employed may be of any conventional composition and may either contain cholesterol or may be cholesterol-free.
• the salt may be substantially entrained within the hydrophobic lipid bilayer which forms the stracture of the liposome.
• the liposomes which are produced may be reduced in size, as through the use of standard sonication and homogenization techniques.
• the liposomal formulations containing the pharmaceutically active compounds identified with the methods described herein may be lyophilized to produce a lyophilizate which may be reconstituted with a phamiaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.
• a phamiaceutically acceptable carrier such as water
• Other pharmaceutical formulations may be prepared from the water- insoluble compounds disclosed herein, or salts thereof, such as aqueous base emulsions.
• the formulation will contain a sufficient amount of pharmaceutically acceptable emulsifying agent to emulsify the desired amount of the compound or salt thereof.
• Particularly useful emulsifying agents include phosphatidyl cholines, and lecithin.
• the pharmaceutical formulations may contain other additives, such as pH-adjusting additives.
• useful pH-adjusting agents include acids, such as hydrochloric acid, bases or buffers, such as sodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodium borate, or sodium gluconate.
• the compositions may contain microbial preservatives.
• Useful microbial preservatives include methylparaben, propylparaben, and benzyl alcohol. The microbial preservative is typically employed when the formulation is placed in a vial designed for multidose use.
• the pharmaceutical formulations of the present invention may be lyophilized using techniques well known in the art.
• any specific pharmacologically active compound identified by methods of the invention will vary somewhat from compound to compound, and subject to subject, and will depend upon the condition of the patient and the route of delivery.
• EXAMPLE 1 Materials and Methods cDNA clones, plasmids constructs and yeast two hybrid assay
• a cDNA sequence encoding full-length mouse cullin 4A was used as a bait to screen a HeLa cell derived cDNA library for cullin-interacting proteins by the yeast two-hybrid assay described in Michel and Xiong, Cell Growth. Differ. 9, 439-445 (1998).
• the full length cDNA clones for both human ROC2 and APCl 1 were isolated by PCR amplification from a HeLa cDNA library and confimied by DNA sequencing. To identify cDNA clones encoding the full length mammalian APC2, the EST database was searched. Full length cDNA clones were not available for human APC2 in the present EST database.
• mouse APC2 EST cDNA clone (W13204) was identified that predicts a 823 amino acid open reading frame with a calculated molecular weight of 94 kDa.
• This mouse cDNA clone is one amino acid residue longer than the published human APC2 (Yu et al., 1998a), but is missing the initiation methionine codon.
• the mouse APC2 was used when testing for the interaction with human APCl 1.
• Yeast cDNA sequences were amplified from yeast genomic DNA by PCR and verified by DNA sequencing.
• the primers used for ScROCl were: 5 '- TTT AAA GAG AAA TAG GAT CCC ATG AGC AAC GAA- 3' [SEQ ID NO: 5] and 5'- TTA AAT GTT TAC GGG GAA TTC ATT TTT TCA CCT-3' [SEQ ID NO: 6] inco ⁇ orating a 5' R ⁇ mHI site and a 3' RcoRI site (underlined) by which the PCR product was inserted in frame into the pGAD prey vector.
• pGBT8- ScROCl was constructed using Smal and Sacl restriction sites from pGEX-
• Primers for amplifying ScAPCll are: 5'-GGC AAT ACA GAT TAG. GAT CCT ATG AAA GTT AAA-3'[SEQ ID NO: 7] and 5'-AAT TGT GAT TTC TAG AAT TCT TTT TTA TCG TAA-3' [SEQ ID NO: 8] incorporating a 5' R ⁇ mHI site and a 3' EcoRI site (underlined) by which the PCR product was inserted in frame into the pGAD vector.
• CDC53 was provided by Dr. Mike Tyers and was subcloned from pMT1144 into pBSKS using BamBI and Notl sites.
• CUL B ORF YGR003w
• ScAPC2 was PCR cloned using primers: 5'- ATC CCC ATG GCT ATG TCA TTT CAG ATT ACC CCA-3'[SEQ ID NO: 13] and 5'-AGC TCG TCG ACA TCA TGA GTT TTT ATG CCC ATT-3' [SEQ ID NO: 14] inco ⁇ orating a 5' Ncol site and a 3' Sail site (underlined) by which the PCR product was inserted in frame into the pGBT8 bait vector.
• Reactions contained 0.1 mM MgCl 2 (Buffer 1), 0.2mM d ⁇ TPs, 0.5 mM each primer and 0.1 mg/ml BSA.
• ScROCl, ScAPCl 1, lROCl and hROC2 were all inserted into the p414-ADH vector (CE ⁇ ) using 5' R ⁇ mHI and 3' -ATzoI restriction sites.
• cD ⁇ A clones For expression in mammalian cells, individual cD ⁇ A clones were subcloned into the pcD ⁇ A3 vector under the control of CMV promoter (Invitrogen), pcDNA3-HA or pcDNA3-Myc, for expressing HA or myc epitope tagged fusion protein.
• CMV promoter Invitrogen
• pcDNA3-HA or pcDNA3-Myc for expressing HA or myc epitope tagged fusion protein.
• yeast two-hybrid assay individual cullin sequences were cloned into pGBT8, a modified version of pGBT9, in frame with the DNA- binding domain of Gal4.
• ROCl, ROC2 and APC 11 were cloned into pGAD in- frame with the DNA activation domain of Gal4.
• DMEM human cervix epithelioid carcinoma
• Saos-2 osteosarcoma
• 293T human transformed primary embryonal kidney c cells.
• Cell transfections were earned out using the LipofectAMINE reagent according to the manufacturer's instmctions (Gibco- BRL). For each transfection, 4 ⁇ g of total plasmid DNA (adjusted with pcDNA3 vector DNAs) was used for 60 mm dish.
• sequence of synthetic peptides used in generating rabbit polyclonal antibodies are as follows: anti-human ROC IN (£MAAAMDVDTPSGTN, amino acid residues 1-14 [SEQ ID NO:15], anti-human ROCIC (CDNREWEFQKYGH, residues 97-108 [SEQ ID NO: 16], anti-human APC 11 (C.RQEWKFKE, residues 76 - 84) [SEQ ID NO: 17], and anti-human CUL2 [CRSQASADEYSYVA, residues 733-745 [SEQ ID NO: 18], See Kipreos et al, 1996, supra; Michel and Xiong, 1998, supra.
• a cysteine (underlined) was added to the N-teiminus of each peptide for covalent coupling of the peptide to activated keyhole limpet haemocyaniii (KLH).
• KLH keyhole limpet haemocyaniii
• All rabbit polyclonal antibodies used in this study were affinity purified using respective peptide columns following the manufacturer's instmction (Sulfolink Kit, Pierce, Rockford, IL). Monoclonal anti- HA (12CA5, Boehringer-Mannheim) and anti-myc (9E10, NeoMarker) antibodies were purchased commercially.
• -Antibody to yeast actin was provided by Dr. J. Pringle. Coupled in vitro transcription and translation reactions were performed using the TNT kit following the manufacturer's instmctions (Promega).
• total lysate was prepared from the HeLa cells pooled from ten 150 mm plates after lysis with the NP-40 lysis buffer and clarified by high speed centrifugation (13,000 g for 30 minutes). Following pre-clearing with uncoated sephadex beads, 100 ⁇ g of affinity purified antibodies to human ROCl was added to the clarified cell lysate. After incubating at 4°C with rotation for 1 hour, protein A beads were added to the lysate and incubated for 1 hour. The beads were washed three times with NP-40 lysis buffer, boiled for 3 minutes in Laemmli loading buffer, and the proteins were separated by SDS-PAGE.
• ROCl -specific associated bands were identified by comparing with a parallel immunoprecipitation of the same HeLa lysate with the same anti-ROCl antibody in the presence of molar excess of competing antigen peptide.
• Competable bands at molecular weight between 70 to 120 kDa were excised from the SDS gel and subjected to in-gel protease digestion using lysylendopeptidase (50 ng / ml).
• Digested peptide fragments was extracted by acetonitrile and separated by reverse-phase high pressure liquid chromatography on a Hewlett Packard 1100 HPLC system using a Cl 8 column (1 mm x 250 mm, Vydac). Protein sequences of individual peptides collected from HPLC were detemiined on an automated ABI microsequencer at Glaxo- Wellcome protein microsequencing facility.
• EXAMPLE 5 Materials and Methods Yeast Strains
• All S. cerevisiae strains were derived from YEF473 (a/ ura3-52/ura3-52 his3 ⁇ - 200/l ⁇ is3 ⁇ -200 t ⁇ l ⁇ -63/t ⁇ l ⁇ -63 leu2 ⁇ -l/leu2 ⁇ -l Iys2-801/lys2-801).
• Yeasts were cultured at 30°C unless otherwise indicated in YP medium or SD medium (lacking appropriate amino acids) containing 2% glucose or 2% raffmose plus varying amounts of galactose, as appropriate. To determine protein expression, yeast cultures were collected by centrifugation, washed once with distill water and stored at -80°C overnight.
• Cell pellets were resuspended in lysis buffer containing 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.2% Triton X-100, ImM DTT, ImM PMSF, ImM NaVO 3 and IX protease inhibitors (25 ⁇ /ml leupeptin, 25 ⁇ /ml aprotonin, ImM benzamidine and lO ⁇ /ml ty ⁇ sin inliibitor). Glass beads were added, and samples were vortexed 4 X 30 sec with at least 30 sec on ice between each vortex. Suspension was transfened to a new Eppendorf tube and centrifuged at 13000g at 4°C for 30 min.
• Protein concentration in the whole cell extract were measured using Broadford assay and equal amount of total protein from each sample was separated by SDS-PAGE and followed by immunoblotting.
• yeast was fixed in 3.7% formaldehyde in culturing medium for llir. in a roller dram at 30°C.
• Mutant yeast strains were constructed using PCR-based gene deletion and modification by homologous recombination according to Longtine et al, Yeast 14(10), 953-961 (1998). Primers for PCR products for all strains constructed were designed based on the sequences published in the database and contained 40 bp of sequence homologous to the gene specific sequence (upper case) and 20 bp homologous to the vector template (lower case). To create a perfect deletion of ROCl and replace it with a module containing the E.
• coli kanr gene (strain JM1), pFA6a-kanMX6 template was used with primers ROC1-F1 (5'- TTCTCCAGTGGCAGAGAACTTTAAAGAGAAATAGTTCAACcggatccccgggtt aa-ttaa 5') [S ⁇ Q ID NO: 19] and ROC1-R1 (5'-
• pFA6a-HA3-TRPl template was used with primers SIC1-F2 (5' CAAGCCAAAGGCATTGTTTCAATCTAGGGAT- CAAGAGCATcggatccccgggttaattaa 3') [SEQ ID NO: 23] and SIC1-R1 (5' TAAAATATAATCGTTCCAGAAA- CTTTTTTTTCATTTCTgaattcgagctcgtttaaac 3') [SEQ ID NO: 24]
• PCR was performed using the Expand Long Template PCR System (Boehringer Mannheim) with the following protocol.
• Mix 1 25 ⁇ l contained 2.5 ⁇ l Expand Buffer 1, 0.8 mM dNTPs, 10 ⁇ g BSA and 2 mM each primer .
• Mix 2 (lOO ⁇ l) contained 7.5 ⁇ l Expand Buffer 1, 0.75 ⁇ L Expand enzyme mixture, and 0.1 ⁇ g template DNA. The two mixes were added together, mixed well and immediately subjected to PCR: 20 cycles of 1 min 94°C, 1 min 55°C, 1 min/kb 68°C followed by a 10 min extension at 68°C.
• PCR products from at least eight reactions were pooled, extracted once with phenolxhlorofo ⁇ n: isoamyl alcohol (25:24:1) and ethanol precipitated.
• PCR products were transfomied into diploid YEF473 yeast (to construct strains JM1 and JM5) or into the haploid strain JM5 (to constmct strain JM7) using a standard protocol and plated onto rich medium (YPD plates for strains JM1 and JM5, and YP plates plus 2% raffmose and 2%. galactose for strain JM7) for two days. Plates were then replica plated onto appropriate selectable medium for 2-3 days. Selected transformants were streaked onto selectable medium twice.
• PCR was perfomied on genomic DNA prepared by lyticase treatment using one primer that annealed to the module integrated and one primer that annealed to a region outside of that altered by the recombination.
• PCR product of the appropriate size confimied homologous recombination.
• 2:2 segregation of the selectable marker also confirmed homologous recombination.
• the innnuno-purified ROC1/CUL1 containing complex immobilized on protein A agarose beads was added to an ubiquitin ligation reaction mixture (30 ⁇ l) that contained 50 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 , 2 mM NaF, 10 nM Okadaic Acid, 2 mM ATP, 0.6 mM DTT, 1 ⁇ g 32P-Ub, 60 ng El and 300 ng mouse CDC34 protein.
• the incubation was at 37°C for 30 min unless otherwise specified herein.
• the reaction mixture was then added to 20 ⁇ l 4X Laemmli loading buffer with 10 mM DDT, and boiled for 3 min prior to 7.5% SDS-PAGE analysis.
• mouse cullin 4A encodes a 759 amino acid protein and shares 96% identity with human CUL4A that was recently identified as a candidate 13q amplicon target gene and was amplified or overexpressed in high percentage of breast cancer samples (Chen et al., 1998, supra). An estimated 3 x 10 6 transformants were screened.
• ROCl regulatory of cullins
• the DNA sequence of ROCl is provided herein in FIG. 2 A as SEQ ID NO: 1.
• ROCl can also interact with cullin 1, 2 and 5 as determined by the yeast two- hybrid assay (Fig. 1A).
• Cullin 3 which interacts with ROCl very weakly in yeast cells was later found to also bind to ROCl in cultured mammalian cells (see below).
• SKPl selectively interacts with CULl only (cf. Michel and Xiong, 1998, supra, FIG. 1A), appears to be a general cullin- interacting protein.
• the mammalian cullin genes encode a family of closely related proteins with molecular weights of approximately 90 kDa.
• CULl interacts with SKPl via an -NH 2 -terminal domain (see Michel and Xiong, 1998, supra).
• ROCl To determine the stmctural basis underlying the specific interaction between cullins and ROCl, the region of CULl required for its interaction with ROCl was mapped.
• a series of CULl deletions from both amino- and carboxyl- terminals fused in- frame with the yeast Gal4 DNA binding domain were tested for their abilities to interact with ROCl in yeast cells.
• ROCl interacts with the C-terminal 527 amino acid residues of CULl, but not the N-terminal 249 residues of CULl (FIG. IB).
• SKPl binds to the N-terminal domain of CULl.
• CULl contains at least two distinct domains, an N-terminal domain for interacting with SKPl and a C-terminal domain for binding with ROCl .
• Such structural separation suggests that ROCl is unlikely to interact with CULl in a competing manner with SKPl .
• ROCl and SKPl may co-exist in the same protein complex with CULl to perform different functions.
• ROCl Represents a Family of RTNG Finger Proteins Related to APCl !
• ROCl encodes an 108 amino acid residue protein with a predicted molecular weight of 12265 D (FIG. 2A, SEQ ID NO: 2).
• Database searches identified ROCl as a highly evolutionarily conserved gene whose S. cerevisiae (ROCl-Sc), S. pombe (ROCl-Sp) and plant (ROCl -At) homologues share 67%, 88% and remarkably 98% protein sequence identity with human ROCl, respectively, over the 82 amino acid region compared (FIG. 2C).
• Database searches have also identified two additional genes, ROC2 in higher eukaryotes and APCl 1 in all eukaryotic species (FIG. 2B and 2C), that are closely related to
• ROCl Human ROC2 and APCll encode an 85 amino acid (Mr. 10007 D) and an 84 residue (Mr. 9805 D) protein, respectively.
• the DNA sequence of ROC2 is provided herein at FIG. 2B as SEQ ID NO:3; its amino acid sequence is provided herein at FIG. 2B as SEQ ID NO:4.
• ROCl and ROC2 share an overall protein sequence identity of 51%o with each other and 38%o and 35% identity with APCl 1, respectively, indicating that ROCl and ROC2 are more closely related to each other than to APCl 1.
• both ROC2 and APCl 1 are also highly conserved during evolution. Therefore, ROC1 ROC2/APC11 define a new family of proteins that are likely to carry out important cellular functions. 38
• ROC/APC11 proteins contain two characteristic features: a RING finger and richness in tryptophan residues.
• the RING finger domain has been found in many eukaryotic proteins with diverse functions and is thought to mediate protein- protein interactions (Borden, K.L. and Freemont, P.S. (1996), Current Opinion in Structural Biology 6, 395-401).
• the majority of RING finger proteins contain a highly conserved structural motif with a histidine residue flanked by three and four cysteine residues on either side (C 3 HC ).
• the ROCl protein from all species has a substitution of the last cysteine with an aspartic acid residue (FIG. 2C).
• the second feature of this family of proteins is six highly conserved tiyptophan residues. Three tryptophan residues in ROCl are followed by an acidic amino acid residue (Asn, Glu or Asp) that resemble the WD repeat and may potentially also be involved in mediating protein-protein interactions.
• APCl 1 was recently identified as a subunit of the yeast APC complex whose loss of function resulted in a defect in the onset of anaphase and exit from mitosis (Zachariae et al., 1998, supra).
• APC2 was found to contain limited sequence similarity to the C-terminal region of cullins. Id.
• Saos-2 cells were transfected with plasmids directing the expression of HA-epitope tagged human ROCl (HA-ROC1) together with CULl or other individual niyc-epitope tagged cullins, as set forth above.
• Transfected cells were metabolically labeled with [ 3D S]-methionine, and cell lysates were immunoprecipitated reciprocally with either anti-HA, anti-CULl or anti-myc antibody (FIG. 3A).
• ROCl e.g., lanes 2 and 3, FIG. 3 A
• HA antibody cross-reacted with the cullins lane 12, FIG.
• cullin 1 or cullin 2 KDVFQK, [SEQ ID NO: 25] conesponding to residues 459-464 in human CULl, database accession AF062536, or 428-433 of human CUL2, accession Q13617
• cullin 2 KIFLENHVRHLH, [SEQ ID NO: 26] residues 62-73, accession Q13617
• cullin 3 KDVFERYY, residues 425-432 [SEQ ID NO: 27]and KVYTYVA, [SEQ ID NO: 28] residues 762-768, accession AF062537
• cullin 4A or 4B KRIESLIDRDY, [SEQ ID NO: 29] residues 396-406 in human CUL4A, accession Q13619 or residues 263-273 in human CUL4B, accession Q13620).
• ROCl was detected in both CULl (lane 1) and CUL2 (lane 5) complexes (lower panel, FIG. 3C). Demonstration of association between ROCl and other cullins by IP-Western was not canied out because of the lack of antibodies to other cullins at present.
• ROC2 and APCl 1 were used to determine whether ROC2 and APCl 1, like ROCl, also interact with cullins.
• Full length human ROC2 or APCl 1 was fused in-frame with the yeast Gal4 DNA activation domain and co-transformed into yeast cells with individual cullins fused to the Gal4 DNA binding domain.
• ROC2 interacted strongly with cullins 1, 2, 4A and 5 (FIG. 4A), indicating that ROC2 is also a general cullin-interacting protein.
• APCl 1 only interacted with cullin 5, but not other cullins (FIG. 4B).
• Saos-2 cells were transfected with plasmids directing the expression of HA tagged human ROC2 (HA-ROC2) or APC11 (HA- APCl 1) together with untagged CULl or individual myc tagged cullins.
• Transfected cells were metabolically labeled with [ 3 S]-methionine, and cell lysates were immunoprecipitated with either anti- HA, anti-CULl or anti-myc antibody (FIG. 4C and 4D).
• Transfected HA-ROC2 protein migrates as a doublet (lanes 6 to 10, FIG. 4C). The myc antibody does not cross-react with either form of ROC2 (e.g. comparing lanes 5 and 6).
• APCl 1 and cullins were not detected to interact with each other in reciprocal precipitations (lane 1 to 10, FIG. 4D).
• Cullin 5 was weakly, but reproducibly, detected in the APCl 1 immunocomplex (lane 10).
• CUL5 is the most divergent member of the cullin family and contains the highest sequence similarity to APC2.
• several cellular proteins including a band of approximately 130 kDa was detected in the ROC2 complex when CUL5, but not other cullins, was co-expressed (FIG. 4C, lanes 5 and 10).
• HeLa cells were transfected with plasmids directing the expression of myc- epitope tagged APC2 with either HA-epitope tagged ROCl, ROC2 or APCl 1, and determined their respective bindings in vivo. Consistent with the yeast two-hybrid assay, APC2 and APCl 1 were reciprocally detected in APCl 1 and APC2 immunocomplexes, respectively (data not shown). Weak binding was detected between ectopically expressed ROCl and APC2, but ROC2, even when ove ⁇ roduced, was not seen to interact with APC2 (data not shown).
• EXAMPLE 12 Decrease of ROClp protein causes a cdc53-. cdc34- and cdc4-like phenotype
• the yeast genome contains a single ROC gene, Sc-ROCl (ORF YOL133w), that shares 67% sequence identity with human ROCl (FIG. 2C), providing a simpler and more genetically facile system to determine the in vivo function of ROC family proteins.
• the consequence of deleting the ScROCl gene by replacing it with a kanamycin resistance module was determined by PCR homologous recombination.
• One copy of ScROCl was replaced in a diploid, and the heterozygous yeast was subjected to spomlation and tetrad dissection (FIG. 5A).
• a conditional yeast strain in which ScROCl was under the control of the galactose-inducible, glucose-repressible GAL1 promoter was created.
• An HA3 tag was fused in- frame with the ScROCl gene to monitor the level of ROCl protein expression.
• Transforaiants were spomlated and dissected (2:2 segregation was observed), and haploid yeast containing GAL-HA3-ScROCl were isolated and verified by PCR analysis (data not shown) and protein expression.(FIG. 5C).
• High levels of expression of HA3-ROClp (FIG. 5B) or untagged ROClp (data not shown) from the GAL1 promoter had no detectable effect on yeast growth.
• ROClp depletion-induced phenotype is indistinguishable from those caused by temperature sensitive mutations in the CDC53, CDC4 and CDC34 genes (Mathias et al., 1996, supra).
• This result suggests that the ScROCl gene is involved in the same pathway as these genes in controlling the ubiquitin-mediated proteo lysis of proteins during the Gl phase of the cell cycle such as CDK inhibitor p40Siclp.
• yeast ROC/APC11 family like their human homologues, could directly interact with the yeast cullin/CDC53 family by the yeast-two-hybrid system.
• the yeast genome contains four cullin members, CDC53, CUL-B (ORF YGR003w), CUL-C (ORF YJL047c) and APC2. Each gene was fused in-frame with the Gal4 DNA binding domain and co-transformed with ScROCl or ScAPCl 1 fused in-frame with the GAL4 activation domain. ScAPC2 was self- activating as a bait and was fused in-framed with the GAL4 activation domain and tested with ScROCl fused in-frame with the DNA binding domain. ScROCl interacted with all four yeast cullin genes including the most distantly related APC2 as determined by the activation of histidine reporter gene.
• ScAPCl 1 only interacted weakly with CUL-C, but not CDC53 or CUL-B (FIG. 5D). Interaction of ScAPCl 1 with ScAPC2 could not be tested because both are self-activating as baits. Hence, like human ROC proteins, yeast ROCl also commonly interacts with all members of cullin family proteins.
• a determination as to whether ScROCl plays a role in regulating protein degradation was based on the phenotypic similarity between ROClp depleted and cdc53 mutant cells and the interaction of ScROCl with CDC53 .
• a critical substrate of the CDC53 pathway is the Gl CDK inhibitor, p40Siclp, which is targeted for ubiquitin mediated degradation by the yeast SCF (Skowyra et al., 1997; Feldman et al, 1997, supra).
• CDC53 the closest yeast homologue of human CULl, assembles into a functional E3 ubiquitin ligase complex in insect cells with E2 CDC34, SKPl and an F box protein (SCF complex) to catalyze ubiquitination of phosphorylated substrates (Skowyra et al., 1997; Feldman et al., 1997, supra).
• Protein complexes containing human CULl, SKPl and SKP2 assembled in insect cells were found to contain little ubiquitin ligase activity, but became active after incubating with HeLa cell lysate (Lyapina et al, 1998, supra), raising the possibility that an additional rate limiting component(s) is required for cullin- dependent ubiquitin ligase activity.
• ROCl may function biochemically as a subunit of ubiquitin ligase activity
• the ubiquitin ligation activity of the ROCl and CULl immunocomplexes was analyzed.
• 293T cells were transiently transfected with plasmid DNA expressing HA epitope tagged ROCl (HA-ROCl) and cullin 1 and ROC1-CUL1 complex was recovered by immunoprecipitation using anti-HA antibody.
• HA-ROCl HA epitope tagged ROCl
• cullin 1 and ROC1-CUL1 complex was recovered by immunoprecipitation using anti-HA antibody.
• the F-box protein SKP2 which has been previously demonstrated to interact with CULl, was included in the transfection.
• SKPl which mediates the binding of CULl with SKP2, is expressed at high level in the cell and was not included in the transfection.
• the ubiquitin ligase activity of ROCl and CULl was measured by incubating the HA-ROC1-CUL1 immunocomplex immobilized on protein A agarose beads with purified human El, mouse E2 CDC34, ATP and 32 P-labeled ubiquitin (Ub). After incubation, the reactions were te ⁇ ninated by boiling the samples in the presence of SDS and reducing agent and mixtures were resolved by SDS-PAGE, followed by autoradiography.
• the observed protein ladder reflects an increment of a single 32P-Ub (-12 kDa in the form of a recombinant protein), a characteristic of ubiquitination reaction.
• the treatment of the reaction mixture with DTT, SDS and boiling significantly reduced, but cannot completely abolish the Ub-El (marked as 32P-Ub-El, FIG. 6) and Ub-CDC34 (marked as 32P-Ub-CDC34) conjugates.
• No exogenous substrate protein was added to the reaction. Accumulation of high molecular weight ubiquitinated proteins could therefore be resulted from either the ubiquitination of a SKP2-targeted substrate(s) co-precipitated with the HA-ROCl complex or a ligation of ubiquitin proteins.
• Such a non-essential role of transfected SKP2 to the ubiquitin ligase activity of the HA-ROCl complex may be due in part to the presence of endogenous SKP2 in 293 cells (Zhang, H., et al, (1995) Cell 82, 915- 925), or indicating a substrate -independent ligation of ubiquitin molecules.
• Omission of CULl severely reduced the ubiquitin ligase activity of the ROCl immunocomplex (lane 4).
• Reciprocally, omission of ROCl from the CULl complex like the omission of CULl from ROCl complex, also significantly reduced ubiquitin ligase activity (comparing lanes 6 and 7).
• ROCl and CULl complexes from either 293T or HeLa cells were immunoprecipitated using affinity purified antibody specific to either protein and assayed for their ability to catalyze ubiquitin ligation (FIG. 6C).
• the ROCl immunocomplex derived from both HeLa (lane 3) and 293T cells (lane 7) actively catalyzed the inco ⁇ oration of 32 P-labeled ubiquitin into high molecular weights in an El (lane 1) and E2 CDC34 (lane 2) dependent manner.
• the CULl complex also exhibited a high level of ubiquitin ligase activity (lane 6).
• the anti-APCll complex exhibited only background levels of ligase activity when similarly incubated with El and E2 CDC34 (lane 5). It has this been determined that the anti-APCl 1 antibody is capable of precipitating APCl 1 as well as a number of additional cellular proteins, likely conesponding to other components of the APC complex (data not shown).
• ROCl and ROC2 interact directly with all five mammalian cullins that we have examined as determined by several different assays both in vitro and in vzvo ' (FIGS. 1 and 3).
• Conventional biochemical purification has further identified ROCl as a stoichiometrically associated subunit of CULl ubiquitin ligase activity (Tan et al, accompanying paper).
• Yeast ROCl is an essential gene whose depletion results in a multiple elongated bud phenotype indistinguishable from that caused by cdc53, cdc34 and cdc4 mutations and results in accumulation of the CDK inhibitor Sicl as in cdc53, cdc34 and cdc4 mutants.
• ROC-related APCl 1 has been shown to be an essential subunit for APC function. Loss of APCl 1 function in yeast resulted in accumulation of APC substrates and caused metaphase anest (Zachariae et al., 1998, supra).
• ROCl is an essential subunit of cullin ubiquitin ligase.
• ROCl and cullin 1 immunocomplexes precipitated from in vivo catalyze ligation of ubiquitins to form polyubiquitin chains.
• Omission of ROCl dramatically reduced ubiquitin ligase activity from the CULl immunocomplex (FIG. 6).
• APCl 1 (ROCl homologue) and APC2 (homologous to cullins) is the ligase in the APC.
• the extensive studies on ubiquitin-mediated proteolysis during the mitotic phase of the cell cycle have identified the APC as the single major E3 ubiquitin ligase required to degrade most mitotic regulatory proteins.
• yeast CDC53 has been identified as a major E3 ligase activity regulating S phase entry. Though the in vivo function of most cullins are yet to be determined, some may well perform other functions unrelated to cell cycle control.

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