WO2001090324A2 - 32263 : une nouvelle enzyme a biotine et ses utilisations - Google Patents

32263 : une nouvelle enzyme a biotine et ses utilisations Download PDF

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Publication number
WO2001090324A2
WO2001090324A2 PCT/US2001/016404 US0116404W WO0190324A2 WO 2001090324 A2 WO2001090324 A2 WO 2001090324A2 US 0116404 W US0116404 W US 0116404W WO 0190324 A2 WO0190324 A2 WO 0190324A2
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bre
nucleic acid
protein
polypeptide
seq
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PCT/US2001/016404
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WO2001090324A3 (fr
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Rachel E. Meyers
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Millennium Pharmaceuticals, Inc.
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Priority to AU2001263342A priority Critical patent/AU2001263342A1/en
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Publication of WO2001090324A3 publication Critical patent/WO2001090324A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • Biotin is an essential water-soluble vitamin of the B-complex group which is synthesized by plants, most prokaryotes and virtually all eukaryotes. Also known as vitamin H, biotin is well characterized in its role as a coenzyme or prosthetic group of a number of enzymes.
  • the biotin group can serve as a carrier of activated CO 2 and is often covalently attached to enzymes at a biotin-attachment domain through the ⁇ -amino group of a ly sine residue.
  • carboxyl group to an acceptor molecule (carboxylase reaction)
  • a reaction which is catalyzed by such biotin enzymes generally occurs in two steps:
  • Biotin enzymes are also involved in the reverse (decarboxylase) reaction.
  • biotin carboxylases and biotin decarboxylases in humans (see, e.g., Knowles (1989) Ann. Rev. Biochem. 58:195-221; Samols et al (1988) J. Biol. Chem. 263:6461-6464).
  • biotin carboxylases are key enzymes in such pathways as gluconeogenesis, lipogenesis, amino acid metabolism, the urea cycle, and energy transduction.
  • Biotin Protein Ligases BPL
  • Biotin Protein Ligases BPL
  • Biotin enzymes play important roles in the synthesis and breakdown of a great number of metabolic intermediates, which may implicate them in a number of pathologies.
  • Several inherited and acquired disorders involving errant biotin metabolism have been described (Baumgartner and Suormala (1997) Int. J. Vitam Nutr Res 67:377-384; Baumgartner and Suormala (1999) Biofactors 10:287-290).
  • These disorders can manifest themselves in a number of symptoms including severe nutritional difficulties, organic aciduria, neurologic abnormalities, and cutaneous distress (rash, alopecia, etc). Accordingly, proteins which are involved with biotin-related metabolism may hold significant therapeutic value.
  • the present invention is based, at least in part, on the discovery of novel members of the family of biotin proteins, referred to herein as Biotin Enzyme- 1 (or BRE) nucleic acid and protein molecules.
  • the BRE nucleic acid and protein molecules of the present invention are useful as modulating agents in regulating a variety of cellular processes, e.g., cellular proliferation, growth, differentiation, protein synthesis, or energy transduction.
  • this invention provides isolated nucleic acid molecules encoding BRE proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of BRE-encoding nucleic acids.
  • a BRE nucleic acid molecule of the invention is at least
  • nucleotide sequence e.g., to the entire length of the nucleotide sequence shown in SEQ ID NO:l or 3, or a complement thereof.
  • the isolated nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO: 1 or 3, or a complement thereof.
  • the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 1-166 of SEQ ID NO: 1.
  • the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 2179-2577 of SEQ ID NO:l.
  • the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO:l or 3.
  • a BRE nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:2.
  • a BRE nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%), 99%o or more identical to the entire length of the amino acid sequence of SEQ ID
  • an isolated nucleic acid molecule encodes the amino acid sequence of human BRE.
  • the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO.
  • the nucleic acid molecule is at least 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-1250, 1250- 1500, 1500-1750, 1750-2000, 2000-2250, 2250-2500 or more nucleotides in length.
  • the nucleic acid molecule is at least 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-1250, 1250-1500, 1500-1750, 1750-2000, 2000- 2250, 2250-2500, or more nucleotides in length and encodes a protein having a BRE activity (as described herein).
  • nucleic acid molecules preferably BRE nucleic acid molecules, which specifically detect BRE nucleic acid molecules relative to nucleic acid molecules encoding non-BRE proteins.
  • a nucleic acid molecule is at least 50-100, 100-250, 250-500, 500- 750, 750-1000, 1000-1250, 1250-1500, 1500-1750, 1750-2000, 2000-2250, 2250-2500 or more nucleotides in length and hybridizes under stringent conditions to a complement of a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:l.
  • the nucleic acid molecules are at least 15 (e.g., 15 contiguous) nucleotides in length and hybridize under stringent conditions to a complement of the nucleotide molecules set forth in SEQ ID NO:l .
  • the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the nucleic acid molecule hybridizes to a complement of a nucleic acid molecule comprising SEQ ID NO:l or 3, respectively, under stringent conditions.
  • Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a BRE nucleic acid molecule, e.g., the coding strand of a BRE nucleic acid molecule.
  • the invention provides a vector comprising a BRE nucleic acid molecule.
  • the vector is a recombinant expression vector.
  • the invention provides a host cell containing a vector of the invention.
  • the invention provides a host cell containing a nucleic acid molecule of the invention.
  • the invention also provides a method for producing a protein, preferably a BRE protein, by culturing in a suitable medium, a host cell, e.g., a mammalian host cell such as a non-human mammalian cell, of the invention containing a recombinant expression vector, such that the protein is produced.
  • an isolated BRE protein includes at least one or more of the following domains: a carbamoyl-phosphate synthase L chain, ATP binding domain (or CPSase domain), and/or a biotin-requiring enzyme domain.
  • a BRE protein includes at least one or more of the following domains: a CPSase domain, a biotin-requiring enzyme domain, and has an amino acid sequence at least about 50%, 55%, 60%, 65%, 67%, 68%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:2.
  • a BRE protein includes at least one or more of the following domains: a CPSase domain, a biotin-requiring enzyme domain and has a BRE activity (as described herein).
  • a BRE protein includes at least one or more of the following domains: a CPSase domain, a biotin-requiring enzyme domain, and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a complement of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l or 3.
  • the invention features fragments of the protein having the amino acid sequence of SEQ ID NO:2, wherein the fragment comprises at least 15 amino acids (e.g., contiguous amino acids) of the amino acid sequence of SEQ ID NO:2.
  • a BRE protein has the amino acid sequence of SEQ ID NO:2.
  • the invention features a BRE protein which is encoded by a nucleic acid molecule consisting of a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%), 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a nucleotide sequence of SEQ ID NO:l or 3, or a complement thereof.
  • This invention further features a BRE protein which is encoded by a nucleic acid molecule consisting of a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l or 3, or a complement thereof.
  • the proteins of the present invention or portions thereof, e.g., biologically active portions thereof, can be operatively linked to a non-BRE polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins.
  • the invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind proteins of the invention, preferably BRE proteins.
  • the BRE proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
  • the present invention provides a method for detecting the presence of a BRE nucleic acid molecule, protein, or polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting a BRE nucleic acid molecule, protein, or polypeptide such that the presence of a BRE nucleic acid molecule, protein or polypeptide is detected in the biological sample.
  • the present invention provides a method for detecting the presence of BRE activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of BRE activity such that the presence of BRE activity is detected in the biological sample.
  • the invention provides a method for modulating BRE activity comprising contacting a cell capable of expressing BRE with an agent that modulates BRE activity such that BRE activity in the cell is modulated.
  • the agent inhibits BRE activity.
  • the agent stimulates BRE activity.
  • the agent is an antibody that specifically binds to a BRE protein.
  • the agent modulates expression of BRE by modulating transcription of a BRE gene or translation of a BRE mRNA.
  • the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of a BRE mRNA or a BRE gene.
  • the methods of the present invention are used to treat a subject having a disorder characterized by aberrant or unwanted BRE protein or nucleic acid expression or activity by administering an agent which is a BRE modulator to the subject.
  • the BRE modulator is a BRE protein.
  • the BRE modulator is a BRE nucleic acid molecule.
  • the BRE modulator is a peptide, peptidomimetic, or other small molecule.
  • the disorder characterized by aberrant or unwanted BRE protein or nucleic acid expression is a BRE-associated disorder (e.g., a carboxylase associated disorder, a decarboxylase-associated disorder).
  • the present invention also provides diagnostic assays for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding a BRE protein; (ii) mis-regulation of the gene; and (iii) aberrant post-translational modification of a BRE protein, wherein a wild- type form of the gene encodes a protein with a BRE activity.
  • the invention provides methods for identifying a compound that binds to or modulates the activity of a BRE protein, by providing an indicator composition comprising a BRE protein having BRE activity, contacting the indicator composition with a test compound, and determining the effect of the test compound on BRE activity in the indicator composition to identify a compound that modulates the activity of a BRE protein.
  • Figures 1A-1C depict the cDNA sequence and predicted amino acid sequence of human BRE (clone Fbh32263).
  • the nucleotide sequence corresponds to nucleic acids 1 to 2577 of SEQ ID NO: 1.
  • the amino acid sequence corresponds to amino acids 1 to 725 of SEQ ID NO: 2.
  • the coding region without the 3' untranslated region of the human BRE gene is shown in SEQ ID NO: 3.
  • Figure 2 depicts a structural, hydrophobicity, and antigenicity analysis of the human Fbh32263 protein.
  • Figure 3A-3C depicts the results of a search which was performed against the
  • Figure 4A-4B depicts an alignment of human BRE (SEQ ID NO:2, depicted as "32263.pro") with known transcarboxylases. These are 3-methylcrotonyl-CoA carboxylase precursor from Arabidopsis (SEQ ID NO:4, GenBank No. AAA67356; depicted as "thai. pro”); a protein similar to propionyl-CoA carboxylase alpha chain from C. elegans (SEQ ID NO:5, GenBank No. AAA93384; depicted as "celegans.pro”); and proprionyl-CoA carboxylase alpha chain precursor from H. sapiens (SEQ ID NO:6, GenBank No. P05165; depicted as "human.pro”).
  • the CPSase domain of the human BRE is indicated in italics.
  • the biotin-requiring enzyme domain of the human BRE is underlined.
  • the alignment was performed using the Clustal algorithm which is part of the MEGALIGN program (e.g., version 3.1.7) which is part of the DNASTAR sequence analysis software package.
  • Biotin Requiring Enzyme or "BRE” nucleic acid and protein molecules, which are novel members of a family of enzymes which possess the ability to associate with biotin molecules (e.g. to associate covalently with a biotin coenzyme, to associate non-covalently with a biotin cofactor) in order to function in their biological capacity (e.g., to convert BRE substrates and metabolites into their corresponding BRE-mediated products).
  • biotin requiring enzyme also called “biotin enzyme”, (referred to herein interchangeably as "BRE"
  • biotin enzyme includes a protein, peptide, or enzyme which is able to interact with one or more molecules of biotin in order to carry out its function(s), e.g., specific reactions in catabolic or anabolic pathways.
  • BRE molecules are involved in the anabolism and catabolism of metabolically important biomolecules, including the metabolism of biochemical molecules necessary for energy production or storage (e.g. , carbohydrate metabolism, lipid metabolism), important cellular metabolites (e.g. amino acids, nucleic acids, urea cycle intermediates), as well as the detoxification (e.g., catabolism) of potentially harmful compounds (e.g., toxins, carcinogens).
  • Examples of BRE molecules include prokaryotic, plant, and mammalian carboxylases, decarboxylases, transcarboxylases, and biotin protein ligases. As biotin enzymes, the BRE molecules of the present invention provide novel diagnostic targets and therapeutic agents to control BRE-associated disorders.
  • BRE proteins comprise a family of BRE molecules.
  • family when referring to the protein and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein.
  • family members can be naturally or non-naturally occurring and can be from either the same or different species.
  • a family can contain a first protein of human origin, as well as other, distinct proteins of human origin or alternatively, can contain homologues of non-human origin, e.g., monkey proteins.
  • Members of a family may also have common functional characteristics.
  • a BRE molecule of the present invention is identified based on the presence of at least one "CPSase” domain in the protein or corresponding nucleic acid molecule.
  • CPSase or “CPSase domain” includes a protein domain having an amino acid sequence of about 64-669 amino acid residues and a bit score of at least 286 when compared against a CPSase domain Markov Model (HMM), e.g., PFAM accession number PF00289.
  • HMM CPSase domain Markov Model
  • a CPSase domain includes a protein domain having an amino acid sequence of about 169-569 amino acid residues and a bit score of at least 386.
  • a CPSase domain includes a protein domain having an amino acid sequence of about 269-469 amino acid residues and a bit score of at least about 486 (e.g., 500, 525, 550, 575, 586.6, 600 or more).
  • a CPSase domain preferably includes a sufficient number of amino acid residues for the enzymatic function of the polypeptide sequence
  • a BRE molecule of the present invention is identified based on the presence of at least one carbamoyl phosphate synthase L chain, N-terminal (“CPSase N-terminal”) domain in the protein or corresponding nucleic acid molecule.
  • CPSase N-terminal domain includes a protein domain having an amino acid sequence of about 100-125 amino acid residues and a bit score of at least 144 when compared against a CPSase domain Markov Model (HMM), e.g. , PFAM accession number PF00289.
  • HMM CPSase domain Markov Model
  • a CPSase N- terminal domain includes a protein domain having an amino acid sequence of about 105- 120 amino acid residues and a bit score of at least 164.
  • a CPSase domain includes a protein domain having an amino acid sequence of about 100-125 amino acid residues (e.g., 113) and a bit score of at least about 184.
  • a BRE molecule of the present invention is identified based on the presence of at least one carbamoyl phosphate synthase ATP-binding ("CPSase ATP-binding") domain in the protein or corresponding nucleic acid molecule.
  • CPSase ATP-binding domain includes a protein domain having an amino acid sequence of about 190-240 amino acid residues and a bit score of at least 190 when compared against a CPSase domain Markov Model (HMM), e.g., PFAM accession number PF02786.
  • HMM CPSase domain Markov Model
  • a CPSase domain includes a protein domain having an amino acid sequence of about 200-230 amino acid residues and a bit score of at least 330.
  • a CPSase domain includes a protein domain having an amino acid sequence of about 210-220 amino acid residues (e.g., 214 amino acid residues) and a bit score of at least about 550 (e.g. , 353 or more).
  • a "carbamoyl-phosphate synthase L chain, ATP binding domain" (“CPSase ATP- binding domain") contains a "carbamoyl phosphate synthase subdomain signature". This domain is implicated in ATP binding and/or catalytic activity.
  • a CPSase domain can include, for example, amino acid residues essential for the enzymatic function of the BRE proteins of the present invention.
  • CPSase domains have been found, for example, in the carbamoylase CPSase (e.g., in duplicate) as well as in a variety of biotin-dependent enzymes (e.g., in single copy) for example acetyl-CoA carboxylase, propionyl-CoA carboxylase, pyruvate carboxylase and urea carboxylase.
  • HMMs Hidden Markov Models
  • the CPSase HMM has been assigned the PFAM Accession PF00289 (http://pfam.wustl.edu), InterPro accession number IPR000901 (http://www.ebi.ac.uk/interpro), and Prosite accession numbers PS00866 and PS00867 (http://www.expasy.ch/prosite).
  • a search was performed against the HMM database using the amino acid sequence (SEQ ID NO:2) of human BRE resulting in the identification of a CPSase domain in the amino acid sequence of human BRE (SEQ ID NO: 2) at about residues 51-419 having a score of 586.6.
  • the results of the search are set forth in Figure 3A-3C.
  • a BRE molecule of the present invention is identified based on the presence of at least one "biotin-requiring enzyme domain" in the protein or corresponding nucleic acid molecule.
  • biotin-requiring enzyme domain includes a protein domain having an amino acid sequence of about 35-95 amino acid residues and a bit score of at least 38 when compared against a biotin- requiring enzyme domain Markov Model (HMM), e.g., PFAM accession number PF00364.
  • HMM biotin-requiring enzyme domain Markov Model
  • a biotin-requiring enzyme domain includes a protein domain having an amino acid sequence of about 45-85 amino acid residues and a bit score of at least 48.
  • a biotin-requiring enzyme domain includes a protein domain having an amino acid sequence of about 55-75 amino acid residues and a bit score of at least 58.
  • a biotin-requiring enzyme domain includes a protein domain having an amino acid sequence of about 60-70 amino acid residues and a bit score of at least about 65 (e.g., 66, 67.8, 69, 70, 75, 100 or more).
  • the biotin requiring enzyme domain binds biotin and contains, or can be characterized by, the presence of a "biotin requiring enzyme attachment site", which itself is characterized by the inclusion of a conserved lysine residue.
  • HMMs Hidden Markov Models
  • the biotin-requiring enzyme domain HMM has been assigned the PFAM Accession PF00364 (http://pfam.wustl.edu), InterPro accession number IPR000089 (http://www.ebi.ac.uk/interpro), and Prosite accession number PS00188 (http://www.expasy.ch/prosite).
  • a search was performed against the HMM database using the amino acid sequence (SEQ ID NO:2) of human BRE resulting in the identification of a biotin-requiring enzyme domain in the amino acid sequence of human BRE (SEQ ID NO: 2) at about residues 650-714 having a score of 67.8.
  • the results of the search are set forth in Figure 3 A.
  • a biotin-requiring enzyme domain as described herein is characterized by the presence of a "biotin-requiring enzyme attachment site.”
  • biotin-requiring enzyme attachment site includes a motif having the consensus sequence [GN]-[DEQTR]-X-[LIVMFY]-X(2)-[LIVM]-X-[AIV]- M-K-[LMAT]- X(3)-[LIVM]-X-[SAV] and is described under Prosite entry number PS00188 (http://www.expasy.ch/prosite).
  • a biotin-requiring enzyme attachment site can be found, for example, within the biotin-requiring enzyme domain of the BRE protein of SEQ ID NO:2 at about residues 671-688.
  • Isolated proteins of the present invention preferably have an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:2, or are encoded by a nucleotide sequence sufficiently identical to SEQ ID NO:l or 3.
  • the term "sufficiently identical” refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity.
  • amino acid or nucleotide sequences which share common structural domains have at least 30%), 40%), or 50%) homology, preferably 60% homology, more preferably 70%-80%, and even more preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as sufficiently identical.
  • amino acid or nucleotide sequences which share at least 30%, 40%>, or 50%), preferably 60%, more preferably 70-80%, or 90-95% homology (and ranges intermediate therein) and share a common functional activity are defined herein as sufficiently identical.
  • a "BRE activity”, “biological activity of BRE” or “functional activity of BRE” refers to an activity exhibited by a BRE protein, polypeptide or nucleic acid molecule (e.g., in a BRE expressing cell or tissue), on a BRE substrate, as determined in vivo, or in vitro, according to standard techniques.
  • a BRE activity is a direct activity, such as processing of a BRE-substrate (e.g., carboxylation, decarboxylation).
  • a "BRE substrate” is a molecule or a metabolite which is processed by a BRE molecule.
  • Exemplary substrates include, but are not limited to, energy metabolites, lipid metabolism intermediates, activated CO 2 , carbonyl groups, urea cycle intermediates, amino acid precursors, and nucleic acid precursors.
  • Examples of BRE substrates also include molecules that are essential for BRE function, e.g., biotin, ATP, acetyl Co A.
  • a BRE activity is an indirect activity, such as a cellular signaling or feedback activity mediated by the processing of a BRE substrate by BRE.
  • the BRE proteins of the present invention have one or more of the following activities: 1) modulate the bioenergetic activities of a cell (e.g., storage or yielding of chemical energy, 2) modulate intra- or intercellular signaling or feedback mechanisms, 3) removal of potentially harmful compounds (e.g., cytotoxic substances) from the cell, or facilitate the neutralization of these molecules through enzymatic alteration (e.g., carboxylation, decarboxylation), 4) modulate the production or breakdown of amino acids or nucleic acids, or modulate the homeostatic balance of available amino acid or nucleic acid pools, 5) specific attachment of biotin to its cognate enzyme, e.g., biotin protein ligase activity.
  • Another embodiment of the invention features isolated BRE proteins and polypeptides having a BRE activity.
  • Other preferred proteins are BRE proteins having one or more of the following domains: a CPSase domain, a biotin- requiring enzyme domain and, preferably, a BRE activity.
  • Additional preferred proteins have at least one CPSase domain, one biotin- requiring enzyme domain, and are, preferably, encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a complement of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l or 3.
  • nucleotide sequence of the isolated human BRE cDNA and the predicted amino acid sequence of the human BRE polypeptide are shown in Figure 1 and in SEQ ID NOs:l and 2, respectively.
  • the human BRE gene which is approximately 2577 nucleotides in length, encodes a protein having a molecular weight of approximately 79.8 kD and which is approximately 725 amino acid residues in length.
  • nucleic acid molecules that encode BRE proteins or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify BRE-encoding nucleic acid molecules (e.g., BRE mRNA) and fragments for use as PCR primers for the amplification or mutation of BRE nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double- stranded, but preferably is double-stranded DNA.
  • isolated nucleic acid molecule includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • isolated includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated.
  • an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated BRE nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 or 3, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or portion of the nucleic acid sequence of SEQ ID NO: 1 or 3 as a hybridization probe, BRE nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • nucleic acid molecule encompassing all or a portion of SEQ ID NO:l or 3 can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NO:l or 3.
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or, alternatively, genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO:l or 3.
  • This cDNA may comprise sequences encoding the human BRE protein (i.e., "the coding region", from nucleotides 165-6599), as well as 5' untranslated sequences (nucleotides 1-166) and 3' untranslated sequences (nucleotides 2179-2577) of SEQ ID NO:l.
  • the nucleic acid molecule can comprise only the coding region of SEQ ID NO:l (e.g., nucleotides 167-2178, corresponding to SEQ ID NO:3).
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO:l or 3, or a portion of any of these nucleotide sequences.
  • a nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NO:l or 3, is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:l or 3, such that it can hybridize to the nucleotide sequence shown in SEQ ID NO:l or 3, respectively, thereby forming a stable duplex.
  • an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least about 50%, 55%, 60%>, 65%, 70%), 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the entire length of the nucleotide sequence shown in SEQ ID NO:l or 3, or a portion of any of these nucleotide sequences.
  • the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO:l or 3, for example, a fragment which can be used as a probe or primer or a fragment encoding a portion of a BRE protein, e.g., a biologically active portion of a BRE protein.
  • the nucleotide sequence determined from the cloning of the BRE gene allows for the generation of probes and primers designed for use in identifying and/or cloning other BRE family members, as well as BRE homologues from other species.
  • the probe/primer typically comprises substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ ID NO:l or 3, of an anti-sense sequence of SEQ ID NO: 1 or 3, or of a naturally occurring allelic variant or mutant of SEQ ID NO:l or 3.
  • a nucleic acid molecule of the present invention comprises a nucleotide sequence which is greater than 50-100, 100-250, 250- 500, 500-750, 750-1000, 1000-1250, 1250-1500, 1500-1750, 1750-2000, 2000-2250, 2250-2500, or more nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NO: 1 or 3.
  • Probes based on the BRE nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co- factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a BRE protein, such as by measuring a level of a BRE-encoding nucleic acid in a sample of cells from a subject e.g., detecting BRE mRNA levels or determining whether a genomic BRE gene has been mutated or deleted.
  • a nucleic acid fragment encoding a "biologically active portion of a BRE protein” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO:l or 3, which encodes a polypeptide having a BRE biological activity (the biological activities of the BRE proteins are described herein), expressing the encoded portion of the BRE protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the BRE protein.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO:l or 3, due to degeneracy of the genetic code and thus encode the same BRE proteins as those encoded by the nucleotide sequence shown in SEQ ID NO: 1 or 3.
  • an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:2.
  • BRE nucleotide sequences shown in SEQ ID NO:l or 3 it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the BRE proteins may exist within a population (e.g., the human population). Such genetic polymorphism in the BRE genes may exist among individuals within a population due to natural allelic variation.
  • the terms "gene” and "recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding a BRE protein, preferably a mammalian BRE protein, and can further include non-coding regulatory sequences, and introns.
  • Allelic variants of human BRE include both functional and non-functional BRE proteins.
  • Functional allelic variants are naturally occurring amino acid sequence variants of the human BRE protein that maintain the ability to process a BRE substrate (e.g., carboxylation, decarboxylation).
  • Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:2, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein.
  • ⁇ on-functional allelic variants are naturally occurring amino acid sequence variants of the human BRE protein that do not have the ability to bind or process a BRE substrate substrate (e.g., carboxylation, decarboxylation), and/or carry out any of the BRE activities described herein.
  • Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:2, or a substitution, insertion or deletion in critical residues or critical regions of the protein.
  • the present invention further provides non-human orthologues of the human BRE protein.
  • Orthologues of the human BRE protein are proteins that are isolated from non-human organisms and possess the same BRE substrate binding and/or modulation of membrane excitability activities of the human BRE protein.
  • Orthologues of the human BRE protein can readily be identified as comprising an amino acid sequence that is substantially identical to SEQ ID NO:2.
  • nucleic acid molecules encoding other BRE family members and, thus, which have a nucleotide sequence which differs from the BRE sequences of SEQ ID NO:l or 3 are intended to be within the scope of the invention.
  • another BRE cDNA can be identified based on the nucleotide sequence of human BRE.
  • nucleic acid molecules encoding BRE proteins from different species, and which, thus, have a nucleotide sequence which differs from the BRE sequences of SEQ ID NO: 1 or 3 are intended to be within the scope of the invention.
  • a mouse BRE cDNA can be identified based on the nucleotide sequence of a human BRE.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of the BRE cDNAs of the invention can be isolated based on their homology to the BRE nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. Nucleic acid molecules corresponding to natural allelic variants and homologues of the BRE cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the BRE gene.
  • an isolated nucleic acid molecule of the invention is at least 15, 20, 25, 30 or more nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l or 3.
  • the nucleic acid is at least 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-1250, 1250-1500, 1500-1750, 1750-2000, 2000- 2250, 2250-2500, or more nucleotides in length.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain hybridized to each other.
  • the conditions are such that sequences at least about 70%>, more preferably at least about 80%), even more preferably at least about 85%> or 90% identical to each other remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, Ausubel et al, eds., John Wiley & Sons, Inc. (1995), sections 2, 4 and 6. Additional stringent conditions can be found in Molecular Cloning: A Laboratory Manual, Sambrook et al.
  • a preferred, non-limiting example of stringent hybridization conditions includes hybridization in 4X sodium chloride/sodium citrate (SSC), at about 65-70°C (or hybridization in 4X SSC plus 50%> formamide at about 42-50°C) followed by one or more washes in IX SSC, at about 65- 70°C.
  • a preferred, non-limiting example of highly stringent hybridization conditions includes hybridization in IX SSC, at about 65-70°C (or hybridization in IX SSC plus 50% formamide at about 42-50°C) followed by one or more washes in 0.3X SSC, at about 65-70°C.
  • a preferred, non-limiting example of reduced stringency hybridization conditions includes hybridization in 4X SSC, at about 50-60°C (or alternatively hybridization in 6X SSC plus 50%. formamide at about 40-45°C) followed by one or more washes in 2X SSC, at about 50-60°C. Ranges intermediate to the above-recited values, e.g., at 65-70°C or at 42-50°C are also intended to be encompassed by the present invention.
  • SSPE lxSSPE is 0.15M NaCl, lOmM ⁇ aH 2 PO 4 , and 1.25mM EDTA, pH 7.4
  • SSC 0.15M NaCl and 15mM sodium citrate
  • T m melting temperature
  • additional reagents may be added to hybridization and/or wash buffers to decrease non-specific hybridization of nucleic acid molecules to membranes, for example, nitrocellulose or nylon membranes, including but not limited to blocking agents (e.g., BSA or salmon or herring sperm carrier DNA), detergents (e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like.
  • an additional preferred, non-limiting example of stringent hybridization conditions is hybridization in 0.25-0.5M NaH 2 PO 4 , 7% SDS at about 65°C, followed by one or more washes at 0.02M NaH 2 PO 4 , 1% SDS at 65°C, see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA 81 :1991-1995, (or alternatively 0.2X SSC, 1% SDS).
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:l or 3 and corresponds to a naturally-occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • allelic variants of the BRE sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO: 1 or 3, thereby leading to changes in the amino acid sequence of the encoded BRE proteins, without altering the functional ability of the BRE proteins.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO:l or 3.
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of BRE (e.g.
  • amino acid residues that are conserved among the BRE proteins of the present invention are predicted to be particularly unamenable to alteration.
  • additional amino acid residues that are conserved between the BRE proteins of the present invention and other members of the BRE family are not likely to be amenable to alteration. Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding BRE proteins that contain changes in amino acid residues that are not essential for activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2.
  • An isolated nucleic acid molecule encoding a BRE protein identical to the protein of SEQ ID NO:2 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:l or 3, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NO:l or 3 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • mutations can be introduced randomly along all or part of a BRE coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for BRE biological activity to identify mutants that retain activity.
  • a mutant BRE protein can be assayed for the ability to metabolize or catabolize biochemical molecules necessary for energy production or storage, permit intra- or intercellular signaling, metabolize or catabolize metabolically important biomolecules (e.g. amino acids, nucleic acids), and to detoxify potentially harmful compounds, or to facilitate the neutralization of these molecules.
  • metabolically important biomolecules e.g. amino acids, nucleic acids
  • an antisense nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire BRE coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding a BRE.
  • coding region refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the coding region of human BRE corresponds to SEQ ID NO:3).
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding BRE.
  • the term “noncoding region” refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of BRE mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of BRE mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of BRE mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • an antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fiuorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5 '-
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a BRE protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules of the invention include direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al (1987) FEBS Lett. 215:327-330).
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave BRE mRNA transcripts to thereby inhibit translation of BRE mRNA.
  • a ribozyme having specificity for a BRE-encoding nucleic acid can be designed based upon the nucleotide sequence of a BRE cDNA disclosed herein (i.e., SEQ ID NO:l or 3).
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a BRE-encoding mRNA. See, e.g., Cech et al. U.S. PatentNo. 4,987,071; and Cech et al. U.S. PatentNo. 5,116,742.
  • BRE mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261 :1411-1418.
  • BRE gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the BRE (e.g., the BRE promoter and/or enhancers; e.g., nucleotides 1-107 of SEQ ID NO:l) to form triple helical structures that prevent transcription of the BRE gene in target cells.
  • nucleotide sequences complementary to the regulatory region of the BRE e.g., the BRE promoter and/or enhancers; e.g., nucleotides 1-107 of SEQ ID NO:l
  • the BRE nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23).
  • peptide nucleic acids refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.
  • PNAs of BRE nucleic acid molecules can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication.
  • PNAs of BRE nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by P ⁇ A- directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., SI nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry- O'Keefe supra).
  • PNAs of BRE can be modified, (e.g., to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of BRE nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNA polymerases), to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B. (1996) supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. (1996) supra and Finn P.J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used as a between the PNA and the 5' end of DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn P.J. et al. (1996) supra).
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser, K.H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et ⁇ /. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648- 652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et ⁇ /. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc.
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549).
  • the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).
  • an endogenous BRE gene within a cell line or microorganism may be modified by inserting a heterologous DNA regulatory element into the genome of a stable cell line or cloned microorganism such that the inserted regulatory element is operatively linked with the endogenous BRE gene.
  • an endogenous BRE gene which is normally "transcriptionally silent", i.e., a BRE gene which is normally not expressed, or is expressed only at very low levels in a cell line or microorganism may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell line or microorganism.
  • a transcriptionally silent, endogenous BRE gene may be activated by insertion of a promiscuous regulatory element that works across cell types.
  • a heterologous regulatory element may be inserted into a stable cell line or cloned microorganism, such that it is operatively linked with an endogenous BRE gene, using techniques, such as targeted homologous recombination, which are well known to those of skill in the art, and described, e.g., in Chappel, U.S. Patent No. 5,272,071; PCT publication No. WO 91/06667, published May 16, 1991.
  • Isolated BRE Proteins and Anti-BRE Antibodies One aspect of the invention pertains to isolated BRE proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-BRE antibodies.
  • native BRE proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • BRE proteins are produced by recombinant DNA techniques.
  • a BRE protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the BRE protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • substantially free of cellular material includes preparations of BRE protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • the language “substantially free of cellular material” includes preparations of BRE protein having less than about 30% (by dry weight) of non-BRE protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-BRE protein, still more preferably less than about 10%) of non-BRE protein, and most preferably less than about 5% non-BRE protein.
  • the BRE protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5%> of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of BRE protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of BRE protein having less than about 30% (by dry weight) of chemical precursors or non-BRE chemicals, more preferably less than about 20% chemical precursors or non-BRE chemicals, still more preferably less than about 10% chemical precursors or non-BRE chemicals, and most preferably less than about 5%> chemical precursors or non-BRE chemicals.
  • a "biologically active portion" of a BRE protein includes a fragment of a BRE protein which participates in an interaction between a BRE molecule and a non-BRE molecule.
  • Biologically active portions of a BRE protein include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the BRE protein, e.g., the amino acid sequence shown in SEQ ID NO:2, which include less amino acids than the full length BRE protein, and exhibit at least one activity of a BRE protein.
  • biologically active portions comprise a domain or motif with at least one activity of the BRE protein, e.g., carboxylase activity, decarboxylase activity, transcarboxylase activity.
  • a biologically active portion of a BRE protein can be a polypeptide which is, for example, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700 or more amino acids in length.
  • Biologically active portions of a BRE protein can be used as targets for developing agents which modulate a BRE mediated activity, e.g., intercellular signaling.
  • a preferred biologically active portion of a BRE protein of the present invention may contain one or more of the following domains: a CPSase domain, and/or a biotin-requiring enzyme domain.
  • other biologically active portions, in which other regions of the protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native BRE protein.
  • the BRE protein has an amino acid sequence shown in SEQ ID NO:2.
  • the BRE protein is substantially identical to SEQ ID NO:2, and retains the functional activity of the protein of SEQ ID NO:2, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above.
  • the BRE protein is a protein which comprises an amino acid sequence at least about 50%, 55%>, 60%, 65%, 70%, 15%, 80%>, 85%, 90%, 95%, 96%, 97%, 98%, 99%) or more identical to SEQ ID NO:2.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%o, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., when aligning a second sequence to .
  • the BRE amino acid sequence of SEQ ID NO:2 having 725 amino acid residues, at least 218, preferably at least 290, more preferably at least 363, even more preferably at least 435, and even more preferably at least 508, 580, 653 or more amino acid residues are aligned).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology").
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J Mol. Biol. (48):444-453 (1970)) algorithm which has been inco ⁇ orated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller ( Comput. Appl Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST See http://www.ncbi.nlm.nih.gov.
  • a BRE “chimeric protein” or “fusion protein” comprises a BRE polypeptide operatively linked to a non-BRE polypeptide.
  • An "BRE polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a BRE molecule
  • a “non-BRE polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the BRE protein, e.g., a protein which is different from the BRE protein and which is derived from the same or a different organism.
  • the BRE polypeptide can correspond to all or a portion of a BRE protein.
  • a BRE fusion protein comprises at least one biologically active portion of a BRE protein. In another preferred embodiment, a BRE fusion protein comprises at least two biologically active portions of a BRE protein.
  • the term "operatively linked" is intended to indicate that the BRE polypeptide and the non-BRE polypeptide are fused in-frame to each other. The non-BRE polypeptide can be fused to the N-terminus or C-terminus of the BRE polypeptide.
  • the fusion protein is a GST-BRE fusion protein in which the BRE sequences are fused to the C-terminus of the GST sequences.
  • Such fusion proteins can facilitate the purification of recombinant BRE.
  • the fusion protein is a BRE protein containing a heterologous signal sequence at its N-terminus.
  • expression and/or secretion of BRE can be increased through use of a heterologous signal sequence.
  • the BRE fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo.
  • the BRE fusion proteins can be used to affect the bioavailability of a BRE substrate.
  • BRE fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a BRE protein; (ii) mis-regulation of the BRE gene; and (iii) aberrant post-translational modification of a BRE protein.
  • the BRE-fusion proteins of the invention can be used as immunogens to produce anti-BRE antibodies in a subject for use in screening assays to identify molecules which inhibit the interaction of BRE with a BRE substrate.
  • a BRE chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, f ⁇ lling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a BRE- encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the BRE protein.
  • the present invention also pertains to variants of the BRE proteins which function as either BRE agonists (mimetics) or as BRE antagonists.
  • Variants of the BRE proteins can be generated by mutagenesis, e.g., discrete point mutation or truncation of a BRE protein.
  • An agonist of the BRE proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a BRE protein.
  • An antagonist of a BRE protein can inhibit one or more of the activities of the naturally occurring form of the BRE protein by, for example, competitively modulating a BRE- mediated activity of a BRE protein.
  • specific biological effects can be elicited by treatment with a variant of limited function.
  • treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the BRE protein.
  • variants of a BRE protein which function as either BRE agonists (mimetics) or as BRE antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a BRE protein for BRE protein agonist or antagonist activity.
  • a variegated library of BRE variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of BRE variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential BRE sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of BRE sequences therein.
  • a degenerate set of potential BRE sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of BRE sequences therein.
  • degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential BRE sequences.
  • Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., ⁇ arang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al (1983) Nucleic Acid Res. 11 :477.
  • libraries of fragments of a BRE protein coding sequence can be used to generate a variegated population of BRE fragments for screening and subsequent selection of variants of a BRE protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a BRE coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the BRE protein.
  • cell based assays can be exploited to analyze a variegated BRE library.
  • a library of expression vectors can be transfected into a cell line, e.g., a neuronal cell line, which ordinarily responds to a BRE ligand in a particular BRE ligand-dependent manner.
  • the transfected cells are then contacted with a BRE ligand and the effect of expression of the mutant on, e.g., membrane excitability of BRE can be detected.
  • Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the BRE ligand, and the individual clones further characterized.
  • An isolated BRE protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind BRE using standard techniques for polyclonal and monoclonal antibody preparation.
  • a full-length BRE protein can be used or, alternatively, the invention provides antigenic peptide fragments of BRE for use as immunogens.
  • the antigenic peptide of BRE comprises at least 8 amino acid residues of ' the amino acid sequence shown in SEQ ID NO:2 and encompasses an epitope of BRE such that an antibody raised against the peptide forms a specific immune complex with the BRE protein.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of BRE that are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity.
  • An BRE immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed BRE protein or a chemically synthesized BRE polypeptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic BRE preparation induces a polyclonal anti-BRE antibody response.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (im unoreacts with) an antigen, such as a BRE.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind BRE molecules.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of BRE.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular BRE protein with which it immunoreacts.
  • Polyclonal anti-BRE antibodies can be prepared as described above by immunizing a suitable subject with a BRE immunogen.
  • the anti-BRE antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized BRE.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against BRE can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J Immunol. 127:539-46; Brown et al. (1980) J Biol. Chem .255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int. J.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the immortal cell line e.g., a myeloma cell line
  • the immortal cell line is derived from the same mammalian species as the lymphocytes.
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines.
  • HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").
  • PEG polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind BRE, e.g. , using a standard ELISA assay.
  • a monoclonal anti-BRE antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with BRE to thereby isolate immunoglobulin library members that bind BRE. Kits for generating and screening phage display libraries are commercially available (e.g. , the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01 ; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S.
  • recombinant anti-BRE antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et /. U.S. PatentNo. 4,816,567; Cabilly et ⁇ /. European Patent Application 125,023; Better et al. (1988) Science
  • An anti-BRE antibody (e.g., monoclonal antibody) can be used to isolate BRE by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti- BRE antibody can facilitate the purification of natural BRE from cells and of recombinantly produced BRE expressed in host cells.
  • an anti-BRE antibody can be used to detect BRE protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the BRE protein.
  • Anti-BRE antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen.
  • Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I,
  • vectors preferably expression vectors, containing a nucleic acid encoding a BRE protein (or a portion thereof).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector is another type of vector, wherein additional DNA segments can be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and "vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adeno viruses and adeno- associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adeno viruses and adeno- associated viruses
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g. , tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., BRE proteins, mutant forms of BRE proteins, fusion proteins, and the like).
  • the recombinant expression vectors of the invention can be designed for expression of BRE proteins in prokaryotic or eukaryotic cells.
  • BRE proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S.
  • fusion proteins can be utilized in BRE activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for BRE proteins, for example.
  • a BRE fusion protein expressed in a retroviral expression vector of the present invention can be utilized to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six (6) weeks).
  • Suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al, (1988) Gene 69:301-315) and pET 1 Id (Studier et al, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET l id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128).
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al, (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the BRE expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari, et al, (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933- 943), pJRY88 (Schultz et al, (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and picZ (InVitrogen Corp, San Diego, CA).
  • BRE proteins can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et ⁇ /. (1987) E Z?OJ. 6:187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, ⁇ . F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1 :268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.
  • promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the ⁇ - fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to BRE mRNA.
  • Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • Another aspect of the invention pertains to host cells into which a BRE nucleic acid molecule of the invention is introduced, e.g., a BRE nucleic acid molecule within a recombinant expression vector or a BRE nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome.
  • the terms "host cell” and "recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a BRE protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a BRE protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a BRE protein.
  • the invention further provides methods for producing a BRE protein using the host cells of the invention.
  • the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding a BRE protein has been introduced) in a suitable medium such that a BRE protein is produced.
  • the method further comprises isolating a BRE protein from the medium or the host cell.
  • the host cells of the invention can also be used to produce non-human transgenic animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which BRE-coding sequences have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous BRE sequences have been introduced into their genome or homologous recombinant animals in which endogenous BRE sequences have been altered.
  • Such animals are useful for studying the function and/or activity of a BRE and for identifying and/or evaluating modulators of BRE activity.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
  • Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous BRE gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • a transgenic animal of the invention can be created by introducing a BRE- encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinj ection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the BRE cDNA sequence of SEQ ID NO:l can be introduced as a transgene into the genome of a non-human animal.
  • a nonhuman homologue of a human BRE gene such as a mouse or rat BRE gene, can be used as a transgene.
  • a BRE gene homologue such as another BRE family member, can be isolated based on hybridization to the BRE cDNA sequences of SEQ ID NO:l or 3, and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to a BRE transgene to direct expression of a BRE protein to particular cells.
  • transgenic founder animal can be identified based upon the presence of a BRE transgene in its genome and/or expression of BRE mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a BRE protein can further be bred to other transgenic animals carrying other transgenes.
  • a vector is prepared which contains at least a portion of a BRE gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the BRE gene.
  • the BRE gene can be a human gene (e.g., the cDNA of SEQ ID NO:3), but more preferably, is a non- human homologue of a human BRE gene (e.g., a cDNA isolated by stringent hybridization with the nucleotide sequence of SEQ ID NO: 1).
  • a mouse BRE gene can be used to construct a homologous recombination nucleic acid molecule, e.g., a vector, suitable for altering an endogenous BRE gene in the mouse genome.
  • the homologous recombination nucleic acid molecule is designed such that, upon homologous recombination, the endogenous BRE gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the homologous recombination nucleic acid molecule can be designed such that, upon homologous recombination, the endogenous BRE gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous BRE protein).
  • the altered portion of the BRE gene is flanked at its 5' and 3' ends by additional nucleic acid sequence of the BRE gene to allow for homologous recombination to occur between the exogenous BRE gene carried by the homologous recombination nucleic acid molecule and an endogenous BRE gene in a cell, e.g., an embryonic stem cell.
  • the additional flanking BRE nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene.
  • homologous recombination nucleic acid molecule typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the homologous recombination nucleic acid molecule (see, e.g., Thomas, K.R. and Capecchi, M. R. (1987) Cell 51:503 for a description of homologous recombination vectors).
  • the homologous recombination nucleic acid molecule is introduced into a cell, e.g., an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced BRE gene has homologously recombined with the endogenous BRE gene are selected (see e.g., Li, E. et ⁇ l (1992) Cell 69:915).
  • the selected cells can then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Ter ⁇ toc ⁇ rcinom ⁇ s and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152).
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
  • transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene.
  • cre/loxP recombinase system of bacteriophage PI.
  • cre/loxP recombinase system of bacteriophage PI.
  • FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al (1991) Science 251 :1351-1355.
  • mice containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385:810-813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669.
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
  • the reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal.
  • the offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
  • compositions suitable for administration can be inco ⁇ orated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be inco ⁇ orated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), fransmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by including in the composition an agent which delays abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by inco ⁇ orating the active compound (e.g., a fragment of a BRE protein or an anti-BRE antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., a fragment of a BRE protein or an anti-BRE antibody
  • dispersions are prepared by inco ⁇ orating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the pu ⁇ ose of oral therapeutic administration, the active compound can be inco ⁇ orated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g. , with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Co ⁇ oration and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50%> of the population) and the ED50 (the dose therapeutically effective in 50%> of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • a therapeutically effective amount of protein or polypeptide ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • an effective dosage ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with antibody, protein, or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • the effective dosage of antibody, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
  • the present invention encompasses agents which modulate expression or activity.
  • An agent may, for example, be a small molecule.
  • small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e,.
  • heteroorganic and organometallic compounds having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. It is understood that appropriate doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher.
  • the dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.
  • Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein.
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • an antibody may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclo hosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.
  • the drug moiety can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic).
  • a BRE protein of the invention has one or more of the following activities: 1) modulation the bioenergetic activities of a cell (e.g., storage or yielding of chemical energy) 2) modulation of intra- or intercellular signaling or feedback mechanisms, 3) removal of potentially harmful compounds (e.g., cytotoxic substances) from the cell, or facilitate the neutralization of these molecules through enzymatic alteration (e.g., carboxylation, decarboxylation), 4) modulation the production or breakdown of amino acids or nucleic acids, or modulate the homeostatic balance of available amino acid or nucleic acid pools, 5) the specific attachment of biotin to its cognate enzyme, e.g. , biotin protein ligase activity.
  • the BRE molecules of the invention are useful for catalyzing carboxylase, decarboxylase, and transcarboxylase reactions.
  • these molecules may be employed in small or large-scale synthesis of either carboxylated moieties or decarboxylated substrate, or in chemical processes that require the production or interconversion of these compounds.
  • Such processes are known in the art (see, e.g., Ullmann et al. (1999) Ullmann's Encyclopedia of Industrial Chemistry, 6th ed. VCH: Weinheim; Gutcho (1983) Chemicals by Fermentation. Park ridge, NJ: Noyes Data Co ⁇ oration (ISBN 0818805086); Rehm et al.
  • the isolated nucleic acid molecules of the invention can be used, for example, to express BRE protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect BRE mRNA (e.g., in a biological sample) or a genetic alteration in a BRE gene, and to modulate BRE activity, as described further below.
  • BRE proteins can be used to treat disorders characterized by insufficient or excessive production of a BRE substrate or production of BRE inhibitors.
  • BRE proteins can be used to screen for naturally occurring BRE substrates, to screen for drugs or compounds which modulate BRE activity, as well as to treat disorders characterized by insufficient or excessive production of BRE protein or production of BRE protein forms which have decreased, aberrant or unwanted activity compared to BRE wild type protein, preferably a BE-associated disorder.
  • a "BE- associated disorder” includes a disorder, disease or condition which is caused or characterized by a misregulation (e.g., downregulation or upregulation) of a biotin enzyme-mediated activity (e.g., BE-mediated activity), for example, carboxylase activity or a decarboxylase activity.
  • Biotin-associated disorders can detrimentally affect cellular functions such as cellular proliferation, growth, differentiation, or migration, cellular regulation of homeostasis, inter- or intra-cellular communication; tissue function, such as cardiac function or musculoskeletal function; systemic responses in an organism, such as nervous system responses, hormonal responses (e.g., insulin response), or immune responses; and protection of cells from toxic compounds (e.g., carcinogens, toxins, mutagens, and toxic byproducts of metabolic activity (e.g., reactive oxygen species)).
  • toxic compounds e.g., carcinogens, toxins, mutagens, and toxic byproducts of metabolic activity (e.g., reactive oxygen species)
  • biotin-associated disorders include CNS disorders such as cognitive and neurodegenerative disorders, examples of which include, but are not limited to, Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, senile dementia, Huntington's disease, Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, and Jakob-Creutzfieldt disease; autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders, such as depression, schizophrenia, schizoaffective disorder, korsakof s psychosis, mania, anxiety disorders, or phobic disorders; learning or memory disorders, e.g., amnesia or age-related memory loss, attention deficit disorder, dysthymic disorder, major depressive disorder, mania, obsessive-compulsive disorder, psychoactive substance use disorders, anxiety, phobias, panic disorder,
  • biotin-associated disorders include cardiac-related disorders.
  • Cardiovascular system disorders in which the BRE molecules of the invention may be directly or indirectly involved include arteriosclerosis, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary artery ligation, vascular heart disease, atrial fibrilation, Jervell syndrome, Lange-Nielsen syndrome, long-QT syndrome, congestive heart failure, sinus node dysfunction, angina, heart failure, hypertension, atrial fibrillation, atrial flutter, dilated cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, coronary artery disease, coronary artery spasm, and arrhythmia.
  • BRE-mediated or related disorders also include disorders of the musculoskeletal system such as paralysis and muscle weakness, e.g., ataxia, myotonia, and myokymia.
  • BRE-associated disorders also include cellular proliferation, growth, differentiation, or migration disorders.
  • Cellular proliferation, growth, differentiation, or migration disorders include those disorders that affect cell proliferation, growth, differentiation, or migration processes.
  • a "cellular proliferation, growth, differentiation, or migration process" is a process by which a cell increases in number, size or content, by which a cell develops a specialized set of characteristics which differ from that of other cells, or by which a cell moves closer to or further from a particular location or stimulus.
  • the BRE molecules of the present invention are involved in signal transduction mechanisms, which are known to be involved in cellular growth, differentiation, and migration processes.
  • the BRE molecules may modulate cellular growth, differentiation, or migration, and may play a role in disorders characterized by aberrantly regulated growth, differentiation, or migration.
  • disorders include cancer, e.g., carcinoma, sarcoma, or leukemia; tumor angiogenesis and metastasis; skeletal dysplasia; hepatic disorders; and hematopoietic and/or myeloproliferative disorders.
  • BRE-associated or related disorders also include hormonal disorders, such as conditions or diseases in which the production and/or regulation of hormones in an organism is aberrant.
  • disorders and diseases include type I and type II diabetes mellitus, pituitary disorders (e.g., growth disorders), thyroid disorders (e.g., hypothyroidism or hyperthyroidism), and reproductive or fertility disorders (e.g. , disorders which affect the organs of the reproductive system, e.g., the prostate gland, the uterus, or the vagina; disorders which involve an imbalance in the levels of a reproductive hormone in a subject; disorders affecting the ability of a subject to reproduce; and disorders affecting secondary sex characteristic development, e.g., adrenal hype ⁇ lasia).
  • BRE-associated or related disorders also include immune disorders, such as autoimmune disorders or immune deficiency disorders, e.g., congenital X-linked infantile hypogammaglobulinemia, transient hypogammaglobulinemia, common variable immunodeficiency, selective IgA deficiency, chronic mucocutaneous candidiasis, or severe combined immunodeficiency.
  • autoimmune disorders or immune deficiency disorders e.g., congenital X-linked infantile hypogammaglobulinemia, transient hypogammaglobulinemia, common variable immunodeficiency, selective IgA deficiency, chronic mucocutaneous candidiasis, or severe combined immunodeficiency.
  • BRE-associated or related disorders also include disorders affecting tissues in which BRE protein is expressed.
  • the invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to BRE proteins, have a stimulatory or inhibitory effect on, for example, BRE expression or BRE activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of BRE substrate.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to BRE proteins, have a stimulatory or inhibitory effect on, for example, BRE expression or BRE activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of BRE substrate.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which
  • BRE protein such techniques can be used to identify ligands for such a protein.
  • a BRE protein modulator can, for example, be used to ameliorate cellular growth or proliferation diseases or disorders, e.g., cancer, or nutritional difficulties, organic aciduria, neurologic abnormalities, and cutaneous distress.
  • Such compounds may include, but are not limited to BRE peptides, anti-BRE antibodies, or small organic or inorganic compounds. Such compounds may also include other cellular proteins or peptides.
  • Compounds identified via assays such as those described herein may be useful, for example, for ameliorating cellular growth and proliferation diseases or disorders.
  • compounds that interact with the BRE protein may include compounds which accentuate or amplify the activity of the bound BRE protein. Such compounds would bring about an effective increase in the level of BRE protein activity, thus ameliorating symptoms.
  • mutations within the BRE gene may cause aberrant types or excessive amounts of BRE proteins to be made which have a deleterious effect that leads to a cellular growth or proliferation disease or disorder.
  • the invention provides assays for screening candidate or test compounds which are substrates of a BRE protein or polypeptide or biologically active portion thereof (e.g. , energy transduction metabolites, urea cycle metabolites, lipid metabolism metabolites, amino acid precursors, nucleic acid precursors).
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a BRE protein or polypeptide or biologically active portion thereof.
  • test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one- compound' library method; and synthetic library methods using affinity chromatography selection.
  • biological libraries are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).
  • an assay is a cell-based assay in which a cell which expresses a BRE protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate BRE activity is determined. Determining the ability of the test compound to modulate BRE activity can be accomplished by monitoring, for example, the production of one or more specific metabolites in a cell which expresses BRE (see, e.g., Saada et al. (2000) Biochem Biophys. Res. Commun. 269: 382-386).
  • the cell for example, can be of mammalian origin, e.g., an epithelial or neuronal cell.
  • the ability of the test compound to modulate BRE binding to a substrate can also be determined. Determining the ability of the test compound to modulate BRE binding to a substrate can be accomplished, for example, by coupling the BRE substrate with a radioisotope or enzymatic label such that binding of the BRE substrate to BRE can be determined by detecting the labeled BRE substrate in a complex.
  • BRE could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate BRE binding to a BRE substrate in a complex. Determining the ability of the test compound to bind BRE can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to BRE can be determined by detecting the labeled compound in a complex.
  • compounds e.g., BRE substrates
  • compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • a microphysiometer can be used to detect the interaction of a compound with BRE without the labeling of either the compound or the BRE. McConnell, H. M. et ⁇ l (1992) Science 257:1906-1912.
  • a "microphysiometer” e.g., Cytosensor
  • LAPS light-addressable potentiometric sensor
  • an assay is a cell-based assay comprising contacting a cell expressing a BRE target molecule (e.g. , a BRE substrate) with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the BRE target molecule. Determining the ability of the test compound to modulate the activity of a BRE target molecule can be accomplished, for example, by determining the ability of the BRE protein to bind to or interact with the BRE target molecule.
  • a BRE target molecule e.g. , a BRE substrate
  • Determining the ability of the BRE protein, or a biologically active fragment thereof, to bind to or interact with a BRE target molecule can be accomplished by one of the methods described above for determining direct binding. In a preferred embodiment, determining the ability of the BRE protein to bind to or interact with a BRE target molecule can be accomplished by determining the activity of the target molecule.
  • the activity of the target molecule can be determined by detecting induction of a cellular response (i.e., cell proliferation, migration and/or metabolic activity), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a target-regulated cellular response.
  • a cellular response i.e., cell proliferation, migration and/or metabolic activity
  • detecting catalytic/enzymatic activity of the target on an appropriate substrate detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase)
  • a target-regulated cellular response i.e., cell proliferation, migration and/or metabolic activity
  • an assay of the present invention is a cell-free assay in which a BRE protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the BRE protein or biologically active portion thereof is determined.
  • Preferred biologically active portions of the BRE proteins to be used in assays of the present invention include fragments which participate in interactions with non-BRE molecules, e.g., fragments with high surface probability scores (see, for example, Figure 2). Binding of the test compound to the BRE protein can be determined either directly or indirectly as described above.
  • the assay includes contacting the BRE protein or biologically active portion thereof with a known compound which binds BRE to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a BRE protein, wherein determining the ability of the test compound to interact with a BRE protein comprises determining the ability of the test compound to preferentially bind to BRE or biologically active portion thereof as compared to the known compound.
  • the assay is a cell-free assay in which a BRE protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the BRE protein or biologically active portion thereof is determined.
  • Determining the ability of the test compound to modulate the activity of a BRE protein can be accomplished, for example, by determining the ability of the BRE protein to bind to a BRE target molecule by one of the methods described above for determining direct binding. Determining the ability of the BRE protein to bind to a BRE target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C.
  • BIOS Biomolecular Interaction Analysis
  • BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g.,
  • BIAcore Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • determining the ability of the test compound to modulate the activity of a BRE protein can be accomplished by determining the ability of the BRE protein to interact with and/or convert a BRE substrate (e.g., to produce a specific metabolite).
  • determining the ability of the test compound to modulate the activity of a BRE protein can be accomplished by determining the ability of the BRE protein to further modulate the activity of a downstream effector of a BRE target molecule.
  • the activity of the effector molecule on an appropriate target can be determined or the binding of the effector to an appropriate target can be determined as previously described.
  • the cell-free assay involves contacting a BRE protein or biologically active portion thereof with a known compound (e.g., a BRE substrate) which binds the BRE protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the BRE protein, wherein determining the ability of the test compound to interact with the BRE protein comprises determining the ability of the BRE protein to preferentially bind to or modulate the activity of a BRE target protein, e.g., catalyze the cleavage, e.g., the hydrolytic cleavage, of a chemical bond within the target protein.
  • a known compound e.g., a BRE substrate
  • determining the ability of the test compound to interact with the BRE protein comprises determining the ability of the BRE protein to preferentially bind to or modulate the activity of a BRE target protein, e.g., catalyze the cleavage, e.g.,
  • Binding of a test compound to a BRE protein, or interaction of a BRE protein with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S-transferase/BRE fusion proteins or glutathione-S- transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or BRE protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of BRE binding or activity determined using standard techniques.
  • BRE protein or a BRE target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated BRE protein or target molecules can be prepared from biotin-NHS (N- hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • biotinylation kit Pierce Chemicals, Rockford, IL
  • streptavidin-coated 96 well plates Piereptavidin-coated 96 well plates
  • antibodies reactive with BRE protein or target molecules but which do not interfere with binding of the BRE protein to its target molecule can be derivatized to the wells of the plate, and unbound target or BRE protein trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the BRE protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the BRE protein or target molecule.
  • modulators of BRE expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of BRE mRNA or protein in the cell is determined.
  • the level of expression of BRE mRNA or protein in the presence of the candidate compound is compared to the level of expression of BRE mRNA or protein in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of BRE expression based on this comparison. For example, when expression of BRE mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of BRE mRNA or protein expression.
  • the candidate compound when expression of BRE mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of BRE mRNA or protein expression.
  • the level of BRE mRNA or protein expression in the cells can be determined by methods described herein for detecting BRE mRNA or protein.
  • the BRE proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al.
  • BRE binding proteins bind to or interact with BRE
  • BRE binding proteins bind to or interact with BRE
  • BRE binding proteins are also likely to be involved in the propagation of signals by the BRE proteins or BRE targets as, for example, downstream elements of a BRE-mediated signaling pathway.
  • BRE binding proteins are likely to be BRE inhibitors.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • the gene that codes for a BRE protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey" or "sample”) is fused to a gene that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the BRE protein.
  • a reporter gene e.g., LacZ
  • the invention pertains to a combination of two or more of the assays described herein.
  • a modulating agent can be identified using a cell- based or a cell free assay, and the ability of the agent to modulate the activity of a BRE protein can be confirmed in vivo, e.g., in an animal such as an animal model for cellular transformation and/or tumorigenesis, or an animal model for a metabolic disorder.
  • This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g. , a BRE modulating agent, an antisense BRE nucleic acid molecule, a BRE-specific antibody, or a BRE binding partner
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein. .
  • the invention features a method of treating a subject having a cellular growth or proliferation disease or disorder that involves administering to the subject a BRE modulator such that treatment occurs.
  • the invention features a method of treating a subject having cancer, e.g., colon cancer or lung cancer, that involves treating a subject with a BRE modulator, such that treatment occurs.
  • Preferred BRE modulators include, but are not limited to, BRE proteins or biologically active fragments, BRE nucleic acid molecules, BRE antibodies, ribozymes, and BRE antisense oligonucleotides designed based on the BRE nucleotide sequences disclosed herein, as well as peptides, organic and non- organic small molecules identified as being capable of modulating BRE expression and/or activity, for example, according to at least one of the screening assays described herein. Any of the compounds, including but not limited to compounds such as those identified in the foregoing assay systems, may be tested for the ability to ameliorate cellular growth or proliferation disease or disorder symptoms. Cell-based and animal model-based assays for the identification of compounds exhibiting such an ability to ameliorate cellular growth or proliferation disease or disorder systems are described herein.
  • cell-based systems may be used to identify compounds which may act to ameliorate cellular growth or proliferation disease or disorder symptoms.
  • such cell systems may be exposed to a compound, suspected of exhibiting an ability to ameliorate cellular growth or proliferation disease or disorder symptoms, at a sufficient concentration and for a time sufficient to elicit such an amelioration of cellular growth or proliferation disease or disorder symptoms in the exposed cells.
  • the cells are examined to determine whether one or more of the cellular growth or proliferation disease or disorder cellular phenotypes has been altered to resemble a more normal or more wild type, non- cellular growth or proliferation disease or disorder phenotype.
  • Cellular phenotypes that are associated with cellular growth and/or proliferation disease states include aberrant proliferation, growth, and migration, anchorage independent growth, and loss of contact inhibition.
  • animal-based cellular growth or proliferation disease or disorder systems may be used to identify compounds capable of ameliorating cellular growth or proliferation disease or disorder symptoms.
  • Such animal models may be used as test substrates for the identification of drugs, pharmaceuticals, therapies, and interventions which may be effective in treating cellular growth or proliferation disorders or diseases.
  • animal models may be exposed to a compound, suspected of exhibiting an ability to cellular growth or proliferation disease or disorder symptoms, at a sufficient concentration and for a time sufficient to elicit such an amelioration of cellular growth or proliferation disease or disorder symptoms in the exposed animals.
  • the response of the animals to the exposure may be monitored by assessing the reversal of disorders or symptoms associated with cellular growth or proliferation disease, for example, reduction in tumor burden, tumor size, and invasive and/or metastatic potential before and after treatment.
  • any treatments which reverse any aspect of cellular growth or proliferation disease or disorder symptoms should be considered as candidates for human cellular growth or proliferation disease or disorder therapeutic intervention.
  • Dosages of test agents may be determined by deriving dose-response curves.
  • Gene expression patterns may be utilized to assess the ability of a compound to ameliorate cellular growth and/or proliferation disease symptoms.
  • the expression pattern of one or more genes may form part of a "gene expression profile” or “transcriptional profile” which may be then be used in such an assessment.
  • “Gene expression profile” or “transcriptional profile”, as used herein, includes the pattern of mRNA expression obtained for a given tissue or cell type under a given set of conditions. Such conditions may include, but are not limited to, cell growth, proliferation, differentiation, transformation, tumorigenesis, metastasis, and carcinogen exposure.
  • Gene expression profiles may be generated, for example, by utilizing a differential display procedure, Northern analysis and/or RT-PCR.
  • BRE gene sequences may be used as probes and/or PCR primers for the generation and corroboration of such gene expression profiles.
  • Gene expression profiles may be characterized for known states within the cell- and/or animal-based model systems. Subsequently, these known gene expression profiles may be compared to ascertain the effect a test compound has to modify such gene expression profiles, and to cause the profile to more closely resemble that of a more desirable profile.
  • administration of a compound may cause the gene expression profile of a cellular growth or proliferation disease or disorder model system to more closely resemble the control system.
  • Administration of a compound may, alternatively, cause the gene expression profile of a control system to begin to mimic a cellular growth and/or proliferation disease state.
  • Such a compound may, for example, be used in further characterizing the compound of interest, or may be used in the generation of additional animal models.
  • cDNA sequences identified herein can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.
  • this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the BRE nucleotide sequences, described herein, can be used to map the location of the BRE genes on a chromosome. The mapping of the BRE sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
  • BRE genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the BRE nucleotide sequences. Computer analysis of the BRE sequences can be used to predict primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the BRE sequences will yield an amplified fragment.
  • Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but human cells can, the one human chromosome that contains the gene encoding the needed enzyme, will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924). Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the BRE nucleotide sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a BRE sequence to its chromosome include in situ hybridization (described in Fan, Y. et al. (1990) Proc. Natl Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step.
  • Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical such as colcemid that disrupts the mitotic spindle.
  • the chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually.
  • the FISH technique can be used with a DNA sequence as short as 500 or 600 bases.
  • clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping pu ⁇ oses. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • differences in the DNA sequences between individuals affected and unaffected with a disease associated with the BRE gene can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymo ⁇ hisms.
  • the BRE sequences of the present invention can also be used to identify individuals from minute biological samples.
  • the United States military, for example, is considering the use of restriction fragment length polymo ⁇ hism (RFLP) for identification of its personnel.
  • RFLP restriction fragment length polymo ⁇ hism
  • an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification.
  • This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult.
  • the sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
  • sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome.
  • the BRE nucleotide sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it. Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
  • the sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue.
  • the BRE nucleotide sequences of the invention uniquely represent portions of the human genome.
  • allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases.
  • Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification pu ⁇ oses. Because greater numbers of polymo ⁇ hisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals.
  • the noncoding sequences of SEQ ID NO:l can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO:3 or 6 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • a panel of reagents from BRE nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual.
  • Using the unique identification database positive identification of the individual, living or dead, can be made from extremely small tissue samples.
  • DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a pe ⁇ etrator of a crime.
  • PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g. , hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.
  • sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual).
  • an "identification marker” i.e. another DNA sequence that is unique to a particular individual.
  • actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments.
  • Sequences targeted to noncoding regions of SEQ ID NO:l are particularly appropriate for this use as greater numbers of polymo ⁇ hisms occur in the noncoding regions, making it easier to differentiate individuals using this technique.
  • polynucleotide reagents include the BRE nucleotide sequences or portions thereof, e.g., fragments derived from the noncoding regions of SEQ ID NO:l having a length of at least 20 bases, preferably at least 30 bases.
  • the BRE nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., thymus or brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such BRE probes can be used to identify tissue by species and/or by organ type.
  • polynucleotide reagents e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., thymus or brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such BRE probes can be used to identify tissue by species and/or by organ type.
  • these reagents e.g., BRE primers or probes can be used to screen tissue culture for contamination (i. e. screen for the presence of a mixture of different types of cells in a culture).
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) pu ⁇ oses to thereby treat an individual prophylactically.
  • diagnostic assays for detennining BRE protein and/or nucleic acid expression as well as BRE activity in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant or unwanted BRE expression or activity.
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with BRE protein, nucleic acid expression or activity. For example, mutations in a BRE gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive pu ⁇ ose to thereby phophylactically treat an individual prior to the onset of a disorder characterized by or associated with BRE protein, nucleic acid expression or activity.
  • Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of BRE in clinical trials.
  • agents e.g., drugs, compounds
  • the present invention encompasses methods for diagnostic and prognostic evaluation of cellular growth or proliferation disorders or diseases, e.g., cancer, including, but not limited to colon cancer and lung cancer, and for the identification of subjects exhibiting a predisposition to such conditions.
  • cancer including, but not limited to colon cancer and lung cancer
  • An exemplary method for detecting the presence or absence of BRE protein or nucleic acid in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting BRE protein or nucleic acid (e.g., mRNA, or genomic DNA) that encodes BRE protein such that the presence of BRE protein or nucleic acid is detected in the biological sample.
  • a compound or an agent capable of detecting BRE protein or nucleic acid e.g., mRNA, or genomic DNA
  • a preferred agent for detecting BRE mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to BRE mRNA or genomic DNA.
  • the nucleic acid probe can be, for example, the BRE nucleic acid set forth in SEQ ID NO:l or 3, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to BRE mRNA or genomic DNA.
  • Other suitable probes for use in the diagnostic assays of the invention are described herein.
  • a preferred agent for detecting BRE protein is an antibody capable of binding to BRE protein, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used.
  • the term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect BRE mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of BRE mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of BRE protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • In vitro techniques for detection of BRE genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of BRE protein include introducing into a subject a labeled anti-BRE antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains protein molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • a preferred biological sample is a serum sample isolated by conventional means from a subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting BRE protein, mRNA, or genomic DNA, such that the presence of BRE protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of BRE protein, mRNA or genomic DNA in the control sample with the presence of BRE protein, mRNA or genomic DNA in the test sample.
  • kits for detecting the presence of BRE in a biological sample can comprise a labeled compound or agent capable of detecting BRE protein or mRNA in a biological sample; means for determining the amount of BRE in the sample; and means for comparing the amount of BRE in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect BRE protein or nucleic acid.
  • increased levels of BRE protein, mRNA or DNA (e.g., cDNA or genomic DNA) in the test sample as compared to the control sample is determinative or predictive of a BRE-related aberrancy (e.g., a cellular growth or proliferation disease or disorder, for example, cancer).
  • a BRE-related aberrancy e.g., a cellular growth or proliferation disease or disorder, for example, cancer.
  • 2-fold levels of expression of BRE in the test sample as compared to the control sample may be determinative or predictive of a BRE-related aberrancy.
  • 5-fold, 10-fold, 100-fold, 500-fold or 1000-fold levels of expression of BRE in the test sample as compared to the control sample may be determinative or predictive of a BRE-related aberrancy.
  • the diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant or unwanted BRE expression or activity.
  • aberrant includes a BRE expression or activity which deviates from the wild type BRE expression or activity.
  • Aberrant expression or activity includes increased or decreased expression or activity, as well as expression or activity which does not follow the wild type developmental pattern of expression or the subcellular pattern of expression.
  • aberrant BRE expression or activity is intended to include the cases in which a mutation in the BRE gene causes the BRE gene to be under-expressed or over-expressed and situations in which such mutations result in a non-functional BRE protein or a protein which does not function in a wild-type fashion, e.g., a protein which does not interact with a BRE substrate, or one which interacts with a non-BRE substrate.
  • the term "unwanted" includes an unwanted phenomenon involved in a biological response such as cellular proliferation.
  • unwanted includes a BRE expression or activity which is undesirable in a subject.
  • the assays described herein can be utilized to identify a subject having or at risk of developing a disorder associated with a misregulation in BRE protein activity or nucleic acid expression, such as a CNS disorder (e.g., a cognitive or neurodegenerative disorder), a cellular proliferation, growth, differentiation, or migration disorder, a cardiovascular disorder, musculoskeletal disorder, an immune disorder, or a hormonal disorder.
  • a CNS disorder e.g., a cognitive or neurodegenerative disorder
  • a cellular proliferation, growth, differentiation, or migration disorder e.g., a cellular proliferation, growth, differentiation, or migration disorder
  • a cardiovascular disorder e.g., a musculoskeletal disorder, an immune disorder, or a hormonal disorder.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing a disorder associated with a misregulation in BRE protein activity or nucleic acid expression, such as a CNS disorder, a cellular proliferation, growth, differentiation, or migration disorder, a musculoskeletal disorder, a cardiovascular disorder, an immune disorder, or a hormonal disorder.
  • a disorder associated with a misregulation in BRE protein activity or nucleic acid expression such as a CNS disorder, a cellular proliferation, growth, differentiation, or migration disorder, a musculoskeletal disorder, a cardiovascular disorder, an immune disorder, or a hormonal disorder.
  • the present invention provides a method for identifying a disease or disorder associated with aberrant or unwanted BRE expression or activity in which a test sample is obtained from a subject and BRE protein or nucleic acid (e.g., mRNA or genomic DNA) is detected, wherein the presence of BRE protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted BRE expression or activity.
  • a test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid (e.g., cerebrospinal fluid or serum), cell sample, or tissue.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant or unwanted BRE expression or activity.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • such methods can be used to determine whether a subject can be effectively treated with an agent for a CNS disorder, a muscular disorder, a cellular proliferation, growth, differentiation, or migration disorder, an
  • the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant or unwanted BRE expression or activity in which a test sample is obtained and BRE protein or nucleic acid expression or activity is detected (e.g. , wherein the abundance of BRE protein or nucleic acid expression or activity is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant or unwanted BRE expression or activity).
  • the methods of the invention can also be used to detect genetic alterations in a BRE gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in BRE protein activity or nucleic acid expression, such as a CNS disorder, a musculoskeletal disorder, a cellular proliferation, growth, differentiation, or migration disorder, a cardiovascular disorder, an immune disorder, or a hormonal disorder.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a BRE-protein, or the mis-expression of the BRE gene.
  • such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a BRE gene; 2) an addition of one or more nucleotides to a BRE gene; 3) a substitution of one or more nucleotides of a BRE gene, 4) a chromosomal rearrangement of a BRE gene; 5) an alteration in the level of a messenger RNA transcript of a BRE gene, 6) aberrant modification of a BRE gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non- wild type splicing pattern of a messenger RNA transcript of a BRE gene, 8) a non- wild type level of a BRE-protein, 9) allelic loss of a BRE gene, and 10) inappropriate post-translational modification of a BRE-protein.
  • assays known in the art which can be used for detecting alterations in a
  • detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077- 1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360-364), the latter of which can be particularly useful for detecting point mutations in a BRE gene (see Abravaya et al.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g. , genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a BRE gene under conditions such that hybridization and amplification of the BRE gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
  • nucleic acid e.g. , genomic, mRNA or both
  • Alternative amplification methods include: self sustained sequence replication (Guatelli, J.C. et al, (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D.Y. et al, (1989) Proc. Natl Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P.M. et al. (1988) Bio-Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
  • mutations in a BRE gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
  • sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes see, for example, U.S. Patent No. 5,498,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations in BRE can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin, M.T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. et al. (1996) Nature Medicine 2: 753- 759).
  • genetic mutations in BRE can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, M.T. et al. supra.
  • a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected.
  • Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the BRE gene and detect mutations by comparing the sequence of the sample BRE with the corresponding wild-type (control) sequence.
  • Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or
  • RNA/RNA or RNA/DNA heteroduplexes Other methods for detecting mutations in the BRE gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242).
  • the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type BRE sequence with potentially mutant RNA or D ⁇ A obtained from a tissue sample.
  • the double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands.
  • RNA DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digesting the mismatched regions.
  • either DNA/DNA or RNA DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol 217:286-295.
  • control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in BRE cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).
  • a probe based on a BRE sequence e.g., a wild-type BRE sequence
  • a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent No. 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in BRE genes.
  • single strand conformation polymo ⁇ hism may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl 9:73-79).
  • Single-stranded DNA fragments of sample and control BRE nucleic acids will be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
  • the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230).
  • Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238).
  • amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • the methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used,. e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a BRE gene.
  • any cell type or tissue in which BRE is expressed may be utilized in the prognostic assays described herein.
  • BRE protein (e.g., the maintainence of cellular homeostasis) can be applied not only in basic drug screening, but also in clinical trials.
  • the effectiveness of an agent determined by a screening assay as described herein to increase BRE gene expression, protein levels, or upregulate BRE activity can be monitored in clinical trials of subjects exhibiting decreased BRE gene expression, protein levels, or downregulated BRE activity.
  • the effectiveness of an agent determined by a screening assay to decrease BRE gene expression, protein levels, or downregulate BRE activity can be monitored in clinical trials of subjects exhibiting increased BRE gene expression, protein levels, or upregulated BRE activity.
  • a BRE gene and preferably, other genes that have been implicated in, for example, a BRE-associated disorder can be used as a "read out" or markers of the phenotype of a particular cell.
  • genes, including BRE that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates BRE activity (e.g., identified in a screening assay as described herein) can be identified.
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of BRE and other genes implicated in the BRE-associated disorder, respectively.
  • the levels of gene expression e.g., a gene expression pattern
  • the levels of gene expression can be quantified by northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of BRE or other genes.
  • the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during treatment of the individual with the agent.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) including the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a BRE protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the BRE protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the BRE protein, mRNA, or genomic DNA in the preadministration sample with the BRE protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly.
  • an agent e.g., an agonist
  • increased administration of the agent may be desirable to increase the expression or activity of BRE to higher levels than detected, i.e., to increase the effectiveness of the agent.
  • decreased administration of the agent may be desirable to decrease expression or activity of BRE to lower levels than detected, i.e. to decrease the effectiveness of the agent.
  • BRE expression or activity may be used as an indicator of the effectiveness of an agent, even in the absence of an observable phenotypic response.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted BRE expression or activity, e.g., a biotin-associated disorder such as a CNS disorder; a cellular proliferation, growth, differentiation, or migration disorder; a, musculoskeletal disorder; a cardiovascular disorder; an immune disorder; or a hormonal disorder.
  • a biotin-associated disorder such as a CNS disorder
  • a cellular proliferation, growth, differentiation, or migration disorder e.g., a cellular proliferation, growth, differentiation, or migration disorder
  • a musculoskeletal disorder
  • cardiovascular disorder e.g., a cellular proliferation, growth, differentiation, or migration disorder
  • an immune disorder e.g., a hormonal disorder.
  • a biotin-associated disorder such as a CNS disorder
  • a cellular proliferation, growth, differentiation, or migration disorder e.g., a, musculoskeletal disorder;
  • the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or "drug response genotype”).
  • a drug response genotype e.g., a patient's "drug response phenotype", or "drug response genotype”
  • another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the BRE molecules of the present invention or BRE modulators according to that individual's drug response genotype.
  • Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.
  • the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted BRE expression or activity, by administering to the subject a BRE or an agent which modulates BRE expression or at least one BRE activity.
  • Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted BRE expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the BRE aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • a BRE, BRE agonist or BRE antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. 2.
  • the modulatory method of the invention involves contacting a cell with a BRE or agent that modulates one or more of the activities of BRE protein activity associated with the cell.
  • An agent that modulates BRE protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring substrate molecule of a BRE protein (e.g., energy transduction metabolites, urea cycle metabolites, lipid metabolism metabolites, amino acid precursors, nucleic acid precursors), a BRE antibody, a BRE agonist or antagonist, a peptidomimetic of a BRE agonist or antagonist, or other small molecule.
  • the agent stimulates one or more BRE activities. Examples of such stimulatory agents include active BRE protein and a nucleic acid molecule encoding BRE that has been introduced into the cell.
  • the agent inhibits one or more BRE activities.
  • inhibitory agents include antisense BRE nucleic acid molecules, anti-BRE antibodies, and BRE inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a BRE protein or nucleic acid molecule.
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) BRE expression or activity.
  • the method involves administering a BRE protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted BRE expression or activity.
  • Stimulation of BRE activity is desirable in situations in which BRE is abnormally downregulated and/or in which increased BRE activity is likely to have a beneficial effect.
  • inhibition of BRE activity is desirable in situations in which BRE is abnormally upregulated and/or in which decreased BRE activity is likely to have a beneficial effect.
  • genes involved in cellular growth or proliferation diseases or disorders may cause such disorders via an increased level of gene activity. In some cases, such up-regulation may have a causative or exacerbating effect on the disease state.
  • a variety of techniques may be used to inhibit the expression, synthesis, or activity of such genes and/or proteins.
  • compounds such as those identified through assays described above, which exhibit inhibitory activity, may be used in accordance with the invention to ameliorate cellular growth or proliferation disease or disorder symptoms.
  • Such molecules may include, but are not limited to, small organic molecules, peptides, antibodies, and the like.
  • compounds can be administered that compete with endogenous ligand for the BRE protein.
  • the resulting reduction in the amount of ligand-bound BRE protein will modulate endothelial cell physiology.
  • Compounds that can be particularly useful for this purpose include, for example, soluble proteins or peptides, such as peptides comprising one or more of the extracellular domains, or portions and/or analogs thereof, of the BRE protein, including, for example, soluble fusion proteins such as Ig- tailed fusion proteins. (For a discussion of the production of Ig-tailed fusion proteins, see, for example, U.S. Pat. No. 5,116,964).
  • compounds such as ligand analogs or antibodies, that bind to the BRE receptor site, but do not activate the protein, (e.g., receptor-ligand antagonists) can be effective in inhibiting BRE protein activity.
  • antisense and ribozyme molecules as described herein, which inhibit expression of the BRE gene may also be used in accordance with the invention to inhibit aberrant BRE gene activity.
  • triple helix molecules may be utilized in inhibiting aberrant BRE gene activity.
  • Antibodies that are both specific for the BRE protein and interfere with its activity may also be used to modulate or inhibit BRE protein function. Such antibodies may be generated using standard techniques described herein, against the BRE protein itself or against peptides corresponding to portions of the protein. Such antibodies include but are not limited to polyclonal, monoclonal, Fab fragments, single chain antibodies, or chimeric antibodies.
  • Lipofectin liposomes may be used to deliver the antibody or a fragment of the Fab region which binds to the target epitope into cells. Where fragments of the antibody are used, the smallest inhibitory fragment which binds to the target protein's binding domain is preferred.
  • peptides having an amino acid sequence corresponding to the domain of the variable region of the antibody that binds to the target gene protein may be used. Such peptides may be synthesized chemically or produced via recombinant DNA technology using methods well known in the art (described in, for example, Creighton (1983), supra; and Sambrook et al. (1989) supra).
  • Single chain neutralizing antibodies which bind to intracellular target gene epitopes may also be administered.
  • Such single chain antibodies may be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population by utilizing, for example, techniques such as those described in Marasco et al. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893).
  • Genes that cause cellular growth or proliferation diseases or disorders may be underexpressed within cellular growth or proliferative situations. Alternatively, the activity of the protein products of such genes may be decreased, leading to the development of cellular growth or proliferation disease or disorder symptoms. Such down-regulation of gene expression or decrease of protein activity might have a causative or exacerbating effect on the disease state.
  • genes that are up-regulated in the disease state might be exerting a protective effect.
  • a variety of techniques may be used to increase the expression, synthesis, or activity of genes and/or proteins that exert a protective effect in response to cellular growth or proliferation disease or disorder conditions.
  • the level BRE activity may be increased to levels wherein cellular growth or proliferation disease or disorder symptoms are ameliorated.
  • the level of BRE activity may be increased, for example, by either increasing the level of BRE gene expression or by increasing the level of active BRE protein which is present.
  • a BRE protein at a level sufficient to ameliorate cellular growth or proliferation disease or disorder symptoms may be administered to a patient exhibiting such symptoms. Any of the techniques discussed below may be used for such administration.
  • One of skill in the art will readily be able to ascertain the concentration of effective, non-toxic doses of the BRE protein, utilizing techniques such as those described above.
  • RNA sequences encoding a BRE protein may be directly administered to a patient exhibiting cellular growth or proliferation disease or disorder symptoms, at a concentration sufficient to produce a level of BRE protein such that cellular growth or proliferation disease or disorder symptoms are ameliorated. Any of the techniques discussed below, which achieve intracellular administration of compounds, such as, for example, liposome administration, may be used for the administration of such RNA molecules.
  • the RNA molecules may be produced, for example, by recombinant techniques such as those described herein.
  • subjects may be treated by gene replacement therapy.
  • One or more copies of a BRE gene, or a portion thereof, that directs the production of a normal BRE protein with BRE function may be inserted into cells using vectors which include, but are not limited to adenovirus, adeno-associated virus, and retrovirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes. Additionally, techniques such as those described above may be used for the introduction of BRE gene sequences into human cells.
  • Cells preferably, autologous cells, containing BRE expressing gene sequences may then be introduced or reintroduced into the subject at positions which allow for the amelioration of cellular growth or proliferation disease or disorder symptoms.
  • Such cell replacement techniques may be preferred, for example, when the gene product is a secreted, extracellular gene product.
  • BRE molecules of the present invention as well as agents, or modulators which have a stimulatory or inhibitory effect on BRE activity (e.g., BRE gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) BRE-associated disorders (e.g., proliferative disorders, CNS disorders, cardiac disorders, metabolic disorders, or muscular disorders) associated with aberrant or unwanted BRE activity.
  • BRE-associated disorders e.g., proliferative disorders, CNS disorders, cardiac disorders, metabolic disorders, or muscular disorders
  • pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a BRE molecule or BRE modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with a BRE molecule or BRE modulator.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp.Pharmacol Physiol. 23(10-11): 983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254- 266.
  • two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymo ⁇ hisms.
  • G6PD glucose-6-phosphate dehydrogenase deficiency
  • oxidant drugs anti-malarials, sulfonamides, analgesics, nitrofurans
  • a genome-wide association relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g. , a "bi-allelic" gene marker map which consists of 60,000-100,000 polymo ⁇ hic or variable sites on the human genome, each of which has two variants.)
  • gene-related markers e.g. , a "bi-allelic” gene marker map which consists of 60,000-100,000 polymo ⁇ hic or variable sites on the human genome, each of which has two variants.
  • Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase II/III drug trial to identify markers associated with a particular observed drug response or side effect.
  • such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymo ⁇ hisms (SNPs) in the human genome.
  • SNP single nucleotide polymo ⁇ hisms
  • a "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA.
  • a SNP may be involved in a disease process, however, the vast majority may not be disease- associated.
  • individuals Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.
  • a method termed the "candidate gene approach” can be utilized to identify genes that predict drug response.
  • a gene that encodes a drug target e.g., a BRE protein of the present invention
  • all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • drug metabolizing enzymes e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19
  • NAT 2 N-acetyltransferase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • the gene coding for CYP2D6 is highly polymo ⁇ hic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6- formed metabolite mo ⁇ hine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to C YP2D6 gene amplification.
  • a method termed the "gene expression profiling" can be utilized to identify genes that predict drug response.
  • a drug e.g., a BRE molecule or BRE modulator of the present invention
  • the gene expression of an animal dosed with a drug can give an indication whether gene pathways related to toxicity have been turned on.
  • Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment of an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a BRE molecule or BRE modulator, such as a modulator identified by one of the exemplary screening assays described herein.
  • the invention is based, at least in part, on the discovery of a human genes encoding a novel protein, referred to herein as BRE.
  • the entire sequence of human clones Fbh32263, was determined and found to contain an open reading frame termed human "BRE", set forth in Figure 1.
  • the amino acid sequence of the human BRE expression product is set forth in Figure 1.
  • the BRE protein sequence set forth in SEQ ID NO:2 comprises about 2577 amino acids and is shown in Figure 1.
  • the coding region (open reading frame) of SEQ ID NO:l is set forth as SEQ ID NO:3.
  • This domain binds biotin and contains, or can be characterized by, the presence of a "biotin requiring enzyme attachment site", which itself is characterized by the inclusion of a conserved lysine residue.
  • This search also resulted in the identification of a "Biotin carboxylase C-terminal domain” in the amino acid sequence of BRE (SEQ ID NO: 2) at about residues 383-490, which is implicated in enzymatic activity.
  • This search also resulted in the identification of a "D-ala D-ala ligase” in the amino acid sequence of BRE (SEQ ID NO:2) at about residues 163-233 (score: 11.1).
  • AAA93384 depicted as “celegans.pro”
  • proprionyl-CoA carboxylase alpha chain precursor from H sapiens SEQ ID NO:6, GenBank No. P05165; depicted as "human.pro”
  • the CPSase domain of the human BRE is indicated in italics.
  • the biotin-requiring enzyme domain of the human BRE. is underlined.
  • BRE is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized.
  • GST glutathione-S-transferase
  • BRE is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199.
  • Expression of the GST-BRE fusion protein in PEB199 is induced with IPTG.
  • the recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined.
  • the pcDNA/Amp vector by Invitrogen Co ⁇ oration (San Diego, CA) is used.
  • This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site.
  • a DNA fragment encoding the entire BRE protein and an ⁇ A tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3' end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.
  • the BRE DNA sequence is amplified by PCR using two primers.
  • the 5' primer contains the restriction site of interest followed by approximately twenty nucleotides of the BRE coding sequence starting from the initiation codon; the 3' end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the BRE coding sequence.
  • the PCR amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, MA).
  • the two restriction sites chosen are different so that the BRE gene is inserted in the correct orientation.
  • the ligation mixture is transformed into E. coli cells (strains HB101, DH5 ⁇ , SURE, available from Stratagene Cloning Systems, La Jolla, CA, can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.
  • COS cells are subsequently transfected with the BRE-pcDNA/Amp plasmid .
  • DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE- dextran-mediated transfection, lipofection, or electroporation.
  • Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the expression of the BRE polypeptide is detected by radiolabelling ( 35 S-methionine or 35 S-cysteine available from NEN, Boston, MA, can be used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988) using an HA specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with 35 S-methionine (or 35 S-cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA-specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.
  • DNA containing the BRE coding sequence is cloned directly into the polylinker of the pCDNA/Amp vector using the appropriate restriction sites.
  • the resulting plasmid is transfected into COS cells in the manner described above, and the expression of the BRE polypeptide is detected by radiolabelling and immunoprecipitation using a BRE specific monoclonal antibody.
  • EXAMPLE 3 TISSUE DISTRIBUTION OF BRE BY TAQMAN
  • BRE mRNA Tissue Expression Analysis of BRE mRNA Using TaqManTM
  • the TaqmanTM procedure is a quantitative, reverse transcription PCR-based approach for detecting mRNA.
  • the RT-PCR reaction exploits the 5' nuclease activity of AmpliTaq GoldTM DNA Polymerase to cleave a TaqManTM probe during PCR.
  • cDNA was generated from the samples of interest, e.g., tumor samples and normal samples, cell lines and the like,and used as the starting material for PCR amplification.
  • a gene-specific oligonucleotide probe (complementary to the region being amplified) was included in the reaction (i.e., the TaqmanTM probe).
  • the TaqManTM probe includes the oligonucleotide with a fluorescent reporter dye covalently linked to the 5' end of the probe (such as FAM (6- carboxyfluorescein), TET (6-carboxy-4,7,2',7'-tetrachlorofluorescein), JOE (6-carboxy- 4,5-dichloro-2,7-dimethoxyfluorescein), or VIC) and a quencher dye (TAMRA (6- carboxy-N,N,N',N'-tetramethylrhodamine) at the 3' end of the probe.
  • a fluorescent reporter dye covalently linked to the 5' end of the probe
  • TET 6-carboxy-4,7,2',7'-tetrachlorofluorescein
  • JOE 6-carboxy
  • the fluorescent signal from the 5' dye is quenched.
  • the 5' to 3' nucleolytic activity of taq polymerase digests the labeled primer, producing a free nucleotide labeled with 6-FAM, which is now detected as a fluorescent signal.
  • the PCR cycle where fluorescence is first released and detected is directly proportional to the starting amount of the gene of interest in the test sample, thus providing a way of quantitating the initial template concentration. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence.
  • the probe specifically anneals between the forward and reverse primer sites.
  • the 5 '-3' nucleolytic activity of the AmpliTaqTM Gold DNA Polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target.
  • the probe fragments are then displaced from the target, and polymerization of the strand continues.
  • the 3' end of the probe is blocked to prevent extension of the probe during PCR. This process occurs in every cycle and does not interfere with the exponential accumulation of product.
  • Samples can be internally controlled by the addition of a second set of primers/probe specific for a housekeeping gene such as ⁇ microglobulin which has been labeled with a different fluor on the 5' end (typically JOE).
  • Table I Expression on BRE in various types of human tissues.
  • Table III Expression on BRE in various types of cancerous and non-cancerous tissues.
  • Table IV Expression of BRE in various cancerous samples.
  • BRE expression levels were measured in various cell types sutiable for animal transplantation by quantitative PCR using the TaqmanTM procedure as described above.
  • the relative levels of BRE expression in various samples is depicted in Table V below.
  • Table V Expression of BRE in a xenograft panel.
  • BRE expression was highest in cells such as breast tumors and breast cancer cell lines and NCIH322. Expression was also noted in colon tumors, ovary and kidney cells .
  • tissues e.g. tissues obtained from normal lung and colon and lung and colon tumors
  • various tissues were first frozen on dry ice.
  • Ten-micrometer-thick sections of the tissues were post-fixed with 4%> formaldehyde in DEPC treated IX phosphate- buffered saline at room temperature for 10 minutes before being rinsed twice in DEPC IX phosphate-buffered saline and once in 0.1 M triethanolamine-HCl (pH 8.0).
  • sections were rinsed in DEPC 2X SSC (IX SSC is 0.15M NaCl plus 0.015M sodium citrate).
  • Tissue was then dehydrated through a series of ethanol washes, incubated in 100%> chloroform for 5 minutes, and then rinsed in 100% ethanol for 1 minute and 95% ethanol for 1 minute and allowed to air dry.
  • Hybridizations were performed with 35 S-radiolabeled (5 X 10 7 cpm/ml) cRNA probes. Probes were incubated in the presence of a solution containing 600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 0.01% sheared salmon sperm DNA, 0.01% yeast tRNA, 0.05% yeast total RNA type XI, IX Denhardt's solution, 50% formamide, 10% dextran sulfate, 100 mM dithiothreitol, 0.1 %> sodium dodecyl sulfate (SDS), and 0.1 %> sodium thiosulfate for 18 hours at 55°C.
  • SDS sodium dodecyl sulfate
  • slides were washed with 2X SSC. Sections are then sequentially incubated at 37°C in TNE (a solution containing 10 mM Tris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA), for 10 minutes, in TNE with lO ⁇ g of RNase A per ml for 30 minutes, and finally in TNE for 10 minutes. Slides were then rinsed with 2X SSC at room temperature, washed with 2X SSC at 50°C for 1 hour, washed with 0.2X SSC at 55°C for 1 hour, and 0.2X SSC at 60°C for 1 hour.
  • TNE a solution containing 10 mM Tris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA
  • Sections were then dehydrated rapidly through serial ethanol-0.3 M sodium acetate concentrations before being air dried and exposed to Kodak Biomax MR scientific imaging film for 24 hours and subsequently dipped in NB-2 photoemulsion and exposed at 4°C for 7 days before being developed and counter stained.
  • the in situ hybridization results essentially agreed with the results of the Taqman analysis.
  • In situ hybridization results a moderate signal in 3 of 4 primary colon tumors, and a strong signal in 3 of 4 colon tumors which had metastasized, e.g. to the liver.
  • Similary BRE expression was undetectable in 1 normal lung sample tested, and moderate in 5 of 6 lung tumors tested. Ovary and breast tumor samples showed no expression. Expression was also strongly detectable in kidney tumors, e.g. Wilm's tumor and renal cell carcinoma.

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Abstract

L'invention concerne de nouvelles molécules d'acide nucléique isolées, appelées molécules d'acide nucléique BRE, qui codent pour de nouvelles molécules apparentées aux enzymes à biotine. L'invention concerne également des molécules d'acide nucléique antisens, des vecteurs d'expression recombinants contenant des molécules d'acide nucléique BRE, des cellules hôtes dans lesquelles on a introduit ces vecteurs d'expression, et des animaux transgéniques dans lesquels on a introduit ou disrupté le gène BRE. L'invention concerne en outre des protéines BRE isolées, des protéines hybrides, des peptides antigéniques, et des anticorps anti-BRE, ainsi que des méthodes diagnostiques faisant appel aux compositions décrites.
PCT/US2001/016404 2000-05-19 2001-05-21 32263 : une nouvelle enzyme a biotine et ses utilisations WO2001090324A2 (fr)

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WO2001007628A2 (fr) * 1999-07-22 2001-02-01 Incyte Genomics, Inc. Synthetases humaines
EP1074617A2 (fr) * 1999-07-29 2001-02-07 Helix Research Institute Amorces pour la synthèse de cADN de pleine longueur et leur utilisation

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