WO2002066626A2 - Regulation de la nucleoside triphosphate pyrophosphohydrolase mut/nudix humaine - Google Patents

Regulation de la nucleoside triphosphate pyrophosphohydrolase mut/nudix humaine Download PDF

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WO2002066626A2
WO2002066626A2 PCT/EP2002/001541 EP0201541W WO02066626A2 WO 2002066626 A2 WO2002066626 A2 WO 2002066626A2 EP 0201541 W EP0201541 W EP 0201541W WO 02066626 A2 WO02066626 A2 WO 02066626A2
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nudix
mut
polypeptide
pyrophosphohydrolase
polynucleotide
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WO2002066626A3 (fr
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Timothy J. Smith
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Bayer Aktiengesellschaft
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the invention relates to the regulation of human MuT/nudix nucleoside triphosphate pyrophosphohydrolase.
  • Proteins of the MutT/nudix family hydrolyze X-lin ed nucleoside diphosphates and, generally, function as "housecleaning" enzyme. There is a need in the art to identify additional members of this family, which can be regulated to provide therapeutic effects.
  • MuT/NUDIX nucleoside trisphosphate pyrophosphohydrolase polypeptide comprising an amino acid sequence selected from the group consisting of:
  • a test compound is contacted with a MuT/NUDIX nucleoside trisphosphate pyrophosphohydrolase polypeptide comprising an amino acid sequence selected from the group consisting of:
  • amino acid sequences which are at least about 37% identical to the amino acid sequence shown in SEQ ID NO: 2 and; the amino acid sequence shown in SEQ ID NO: 2;
  • Binding between the test compound and the MuT/NUDLX nucleoside trisphosphate pyrophosphohydrolase polypeptide is detected.
  • a test compound which binds to the MuT/NUDLX nucleoside trisphosphate pyrophosphohydrolase polypeptide is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • the agent can work by decreasing the activity of the MuT/NUDLX nucleoside tris- phosphate pyrophosphohydrolase.
  • Another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a polynucleotide encoding a MuT/NUDIX nucleoside trisphosphate pyrophospho- hydrolase polypeptide, wherem the polynucleotide comprises a nucleotide sequence selected from the group consisting of:
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1 and; the nucleotide sequence shown in SEQ ID NO: 1.
  • a test compound which binds to the polynucleotide is identified as a potential agent for decreasing extracellular matrix degradation.
  • the agent can work by decreasing the amount of the MuT/NUDIX nucleoside trisphosphate pyrophosphohydrolase through interacting with the MuT/NTJDLX nucleoside trisphosphate pyrophosphohydrolase RNA.
  • Another embodiment of the invention is a method of screening for agents which regulate extracellular matrix degradation.
  • a test compound is contacted with a MuT/NUDIX nucleoside trisphosphate pyrophosphohydrolase polypeptide com- prising an amino acid sequence selected from the group consisting of:
  • amino acid sequences which are at least about 37% identical to the amino acid sequence shown in SEQ ID NO: 2 and; the amino acid sequence shown in SEQ ID NO: 2;
  • a MuT/NUDIX nucleoside trisphosphate pyrophosphohydrolase activity of the polypeptide is detected.
  • a test compound which increases MuT/NUDIX nucleoside trisphosphate pyrophosphohydrolase activity of the polypeptide relative to MuT NUDIX nucleoside trisphosphate pyrophosphohydrolase activity in the absence of the test compound is thereby identified as a potential agent for increasing extracellular matrix degradation.
  • a test compound which decreases MuT/NUDLX nucleoside trisphosphate pyrophosphohydrolase activity of the polypeptide relative to MuT/NUDIX nucleoside trisphosphate pyrophosphohydrolase activity in the absence of the test compound is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • a test compound is contacted with a MuT/NUDLX nucleoside trisphosphate pyrophosphohydrolase product of a poly- nucleotide which comprises a nucleotide sequence selected from the group consisting of:
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1 and; the nucleotide sequence shown in SEQ ID NO: 1 ; Binding of the test compound to the MuT/NUDIX nucleoside trisphosphate pyrophosphohydrolase product is detected.
  • a test compound which binds to the MuT/NUDLX nucleoside trisphosphate pyrophosphohydrolase product is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • Still another embodiment of the invention is a method of reducing extracellular matrix degradation.
  • a cell is contacted with a reagent which specifically binds to a polynucleotide encoding a MuT/NUDIX nucleoside trisphosphate pyrophosphohydrolase polypeptide or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1 and; the nucleotide sequence shown in SEQ TD NO: 1 ;
  • MuT/NUDLX nucleoside trisphosphate pyrophosphohydrolase activity in the cell is thereby decreased.
  • the invention thus provides a human MuT/nudix nucleoside triphosphate pyrophosphohydrolase that can be used to identify test compounds that may act, for example, as activators or inhibitors at the enzyme's active site.
  • Human MuT/nudix nucleoside triphosphate pyrophosphohydrolase and fragments thereof also are useful in raising specific antibodies that can block the enzyme and effectively reduce its activity.
  • Fig. 1 shows the DNA-sequence encoding a MuT/NUDLX nucleoside trisphos- phate pyrophosphohydrolase Polypeptide (SEQ ID NO: 1).
  • Fig. 2 shows the amino acid sequence deduced from the DNA-sequence of Fig.1 (SEQ ID NO: 2).
  • Fig. 3 shows the DNA-sequence encoding a MuT/NUDLX nucleoside trisphos- phate pyrophosphohydrolase Polypeptide (SEQ ID NO: 3).
  • Fig. 4 shows the DNA-sequence encoding a MuT/NUDLX nucleoside trisphosphate pyrophosphohydrolase Polypeptide (SEQ ID NO: 4).
  • Fig. 5 shows the DNA-sequence encoding a MuT/NUDLX nucleoside trisphosphate pyrophosphohydrolase Polypeptide (SEQ ID NO: 5).
  • Fig. 6 shows the DNA-sequence encoding a MuT/NUDLX nucleoside trisphosphate pyrophosphohydrolase Polypeptide (SEQ ID NO: 6).
  • Fig. 7 shows the DNA-sequence encoding a MuT/NUDIX nucleoside trisphosphate pyrophosphohydrolase Polypeptide (SEQ ID NO: 7).
  • Fig. 8 shows the DNA-sequence encoding a MuT/NUDIX nucleoside trisphos- phate pyrophosphohydrolase Polypeptide (SEQ ID NO: 8).
  • Fig. 9 shows the BLASTP - alignment of SEQ ID NO: 2 against trembl
  • Fig. 10 shows the Alignment of MuT/nudix nucleoside triphosphate pyrophosphohydrolase with genomic DNA.
  • the invention relates to an isolated polynucleotide from the group consisting of:
  • amino acid sequences which are at least about 37% identical to the amino acid sequence shown in SEQ ID NO: 2 and; the amino acid sequence shown in SEQ ID NO: 2; ' '
  • e a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d) and encodes a MuT/nudix nucleoside trisphosphate pyrophosphohydrolase polypeptide.
  • a novel MuT/nudix nucleoside triphosphate pyrophosphohydrolase can be used in therapeutic methods to treat cancer, a cardiovascular disorder or a CNS disorder.
  • the MutT or "Nudix" hydrolase family of enzymes hydrolyzes a nucleoside (N) diphosphate (p2) connected to some other moiety X, thus the suggested mnemonic "nudix.” 1.
  • the first MutT was discovered in bacteria.
  • the bacterial mutT protein is involved in the GO system and is responsible for removing an oxidatively damaged and mutagenic form of guanine (8-hydroxyguanine or 7,8-dihydro-8-oxoguanine) from DNA and the nucleotide pool.
  • 8-oxo-dGTP is inserted opposite to dA and dC residues of template DNA with almost equal efficiency, thus leading to A.T to G C trans versions.
  • MutT specifically degrades 8-oxo-dGTP to the monophosphate with the concomitant release of pyrophosphate.
  • MutT is a small protein of about 12 to 15
  • MutT contains a signature motif that studies indicate is the catalytic domain for the hydrolysis of a nucleoside triphosphate.
  • This motif, G-x(5)-E-x(4)-[STAGC]- [LINMA]-x-R-E-[LIVMF]-x-E-E is highly conserved among all members of the MutT/nudix family of proteins (1).
  • the catalytic activity of this domain is not limited to the hydrolysis of nucleoside triphosphates.
  • Orfl68- Ap3A + H2O-ADP + AMP Ap4-tetraphosphatase- Ap4A + H2O--ATP + AMP (purified from human placenta and cloned).
  • MutT/nudix signature motif all of these enzymes share the MutT/nudix signature motif and they all hydrolyze X- linked nucleoside diphosphates.
  • substrates for this family of enzymes vary widely — ucleoside triphosphates, coenzymes, nucleotide sugars, and dinucleo- tide polyphosphates (1).
  • Bessman et al. (1) categorize the MutT/nudix family of proteins as "housecleaning" genes that rid the cell of potentially toxic metabolites and to modulate the accumulation of intermediates in various biochemical pathways.
  • Human MuT/nudix nucleoside triphosphate pyrophosphohydrolase comprises the amino acid sequence shown in SEQ ID NO: 2.
  • a coding sequence for human MuT/nudix nucleoside triphosphate pyrophosphohydrolase is shown in SEQ ID NO: 1. This sequence is located on chromosome 2.
  • Related ESTs (SEQ ID NOS: 4-8) are expressed in kidney, adenocarcinoma, pineal body, ovary, and cerebral cortex. This expression pattern indicates a potential role for this protein in cancer and CNS.
  • BLOCKS, HMMPFAM, and Prosite searches identified the MutT/nudix signature domain in SEQ ID NO: 2, which is the catalytic domain.
  • BLAST alignment shows 41%o identity to a "MutT/nudix family protein" from Deinococcus radiodurans. (FIG. 1).
  • the conserved domain shown in FIG. 1 in bold) could be involved in the active center of a family of pyrophosphate-releasing NTPases.
  • the core region of the domain was selected; it contains four conserved glutamate residues: -Consensus pattern: G-x(5)-E-x(4)-[STAGC]-[LTVMA]-x-R-E- [LIVMF]-x-E-E.
  • Human MuT/nudix nucleoside triphosphate pyrophosphohydrolase of the invention is expected to be useful for the same purposes as previously identified MuT/nudix nucleoside triphosphate pyrophosphohydrolase enzymes.
  • Human MuT/nudix nucleoside triphosphate pyrophosphohydrolase is believed to be useful in therapeutic methods to treat disorders such as cancer, cardiovascular disorders, and CNS disorders.
  • Human MuT/nudix nucleoside triphosphate pyrophosphohydrolase also can be used to screen for human MuT/nudix nucleoside triphosphate pyrophosphohydrolase activators and inhibitors.
  • Human MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides comprise at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150,
  • a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide of the invention therefore can be a portion of a MuT/nudix nucleoside triphosphate pyro- phosphohydrolase protein, a full-length MuT/nudix nucleoside triphosphate pyrophosphohydrolase protein, or a fusion protein comprising all or a portion of a MuT/nudix nucleoside triphosphate pyrophosphohydrolase protein.
  • MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide variants have amino acid sequences which are at least about 37, 40, 45, 50, 55, 60, 65, or 70, preferably about 75, 80, 85, 90, 96, 96, 98, or 99% identical to the amino acid sequence shown in SEQ ID NO: 2 or a fragment thereof. Percent identity between a putative MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide variant and an amino acid sequence of SEQ ID NO: 2 is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio.
  • the trimmed initial regions are examined to determine whether the regions can be joined to for man approximate alignment with gaps.
  • the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch- Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allows for amino acid insertions and deletions.
  • FASTA FASTA program by modifying the scoring matrix file ("SMATRLX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above.
  • the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default.
  • Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions.
  • Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can be found using computer programs well known in the art, such as DNASTAR software. Whether an amino acid change results in a biologically active MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can • readily be determined by assaying for MuT/nudix nucleoside triphosphate pyrophosphohydrolase activity, as described for example, in Bhatnagar, S. K., Bessman, M. J. (1988) J. Biol. Chem. 263, 8953-8957; Kamath, A.
  • Fusion proteins are useful for generating antibodies against MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins that interact with portions of a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
  • a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond.
  • the first polypeptide segment comprises at least 6, 10, 15, 20, 25, 50, 75, 100,
  • the first polypeptide segment also can comprise full-length MuT/nudix nucleoside triphosphate pyrophosphohydrolase protein.
  • the second polypeptide segment can be a full-length protein or a protein fragment.
  • Proteins commonly used in fusion protein construction include ⁇ -galactosidase, ⁇ - glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horse- radish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).
  • epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.
  • a fusion protein also can be engineered to contain a cleavage site located between the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide-encoding sequence and the heterologous protein sequence, so that the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can be cleaved and purified away from the heterologous moiety.
  • a fusion protein can be synthesized chemically, as is known in the art.
  • a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology.
  • Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from SEQ ID NO: 1 in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art.
  • Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA), Santa Cruz Biotechnology (Santa Cruz, CA), MBL International Corporation (MIC;
  • Species homologs of human MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can be obtained using MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide polynucleotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide, and expressing the cDNAs as is known in the art.
  • a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide.
  • a coding sequence for human MuT/nudix nucleoside triphosphate pyrophosphohydrolase is shown in SEQ ID NO: 1.
  • nucleotide sequences encoding human MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides, as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, 98, or 99%o identical to the nucleotide sequence shown in SEQ ID NO: 1 or its complement also are MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotides.
  • Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affme gap search with a gap open penalty of -12 and a gap extension penalty of -2.
  • Complementary DNA (cDNA) molecules, species ho- mologs, and variants of MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotides that encode biologically active MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides also are MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotides.
  • Polynucleotide fragments comprising at least 8, 9, 10, 11, 12, 15, 20, or 25 contiguous nucleotides of SEQ ID NO: 1 or its complement also are MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotides. These fragments can be used, for example, as hybridization probes or as antisense oligonucleotides.
  • MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotides described above also are MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotides.
  • homologous MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotides under stringent conditions, as is known in the art.
  • homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.
  • Species homologs of the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast.
  • Human variants of MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T m of a double-stranded DNA decreases by 1-1.5°C with every 1% decrease in homology (Bonner et al, J. Mol.
  • Variants of human MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotides or MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotides of other species can therefore be identified by hybridizing a putative homologous MuT/nudix nucleoside triphosphate pyrophos- phohydrolase polynucleotide with a polynucleotide having a nucleotide sequence of
  • test hybrid SEQ ID NO: 1 or the complement thereof to form a test hybrid.
  • the melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.
  • Nucleotide sequences which hybridize to MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotides or their complements following stringent hybridization and/or wash conditions also are MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotides.
  • Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.
  • T m of a hybrid between a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotide having a nucleotide sequence shown in SEQ ID NO: 1 or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):
  • Stringent wash conditions include, for example, 4X SSC at 65°C, or 50% formamide,
  • a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids.
  • Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotides.
  • restriction enzymes and probes can be used to isolate polynucleotide fragments, which comprise MuT/nudix nucleoside triphosphate pyrophosphohydrolase nucleotide sequences.
  • Isolated polynucleotides are in preparations that are free or at least 70, 80, or 90%) free of other molecules.
  • Human MuT/nudix nucleoside triphosphate pyrophosphohydrolase cDNA molecules can be made with standard molecular biology techniques, using MuT/nudix nucleoside triphosphate pyrophosphohydrolase mRNA as a template. Human MuT/nudix nucleoside triphosphate pyrophosphohydrolase cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.
  • MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotides can be synthesized using synthetic chemistry techniques to synthesize MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotides.
  • the degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide having, for example, an amino acid sequence shown in SEQ ID NO: 2 or a biologically active variant thereof.
  • PCR-based methods can be used to extend the nucleic acid sequences disclosed herein to detect upstream sequences such as promoters and regulatory elements.
  • restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al, Nucleic Acids Res. 16, 8186, 1988).
  • Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72°C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • capture PCR involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al, PCR Methods Applic. 1, 111-119, 1991).
  • multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
  • Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products.
  • capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) that are laser activated, and detection of the emitted wavelengths by a charge coupled device camera.
  • Output/light intensity can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled.
  • Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA that might be present in limited amounts in a particular sample.
  • Human MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides can be obtained, for example, by purification from human cells, by expression of MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotides, or by direct chemical synthesis.
  • Human MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides can be purified from any cell that expresses the polypeptide, including host cells that have been transfected with MuT/nudix nucleoside triphosphate pyrophosphohydrolase expression constructs.
  • a purified MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide is separated from other compounds that normally associate with the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art.
  • Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.
  • a preparation of purified MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS- polyacrylamide gel electrophoresis.
  • the polynucleotide can be inserted into an expression vector that contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1989.
  • a variety of expression vector/host systems can be utilized to contain and express sequences encoding a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.
  • control elements or regulatory sequences are those non-translated regions of the vector ⁇ enhancers, promoters, 5' and 3' untranslated regions — which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the
  • BLUESCRTPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used.
  • the baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable.
  • vectors based on SV40 or EBV can be used with an appropriate selectable marker.
  • a number of expression vectors can be selected depending upon the use intended for the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide.
  • vectors which direct high level expression of fusion proteins that are readily purified can be used.
  • Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUES CRIPT (Stratagene).
  • a sequence encoding the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ - galactosidase so that a hybrid protein is produced.
  • pTJNT vectors Van Heeke & Schuster, J. Biol. Chem. 264, 5503-5509, 1989
  • pGEX vectors Promega, Madison, Wis.
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH.
  • sequences encoding MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides can be driven by any of a number of promoters.
  • viral promoters such as the
  • 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6, 307-311, 1987).
  • plant promoters such as the small subunit of RUBIS CO or heat shock promoters can be used (Coruzzi et al, EMBO J. 3, 1671-1680, 1984; Broglie et al,
  • constructs can be introduced into plant cells by direct DNA transformation or by pathogen mediated transfection.
  • pathogen mediated transfection Such techniques are described in a number of generally available reviews (e.g., Hobbs or Murray, in MCGRAW HILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y., pp.
  • An insect system also can be used to express a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • Sequences encoding MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.
  • MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides can be expressed (Engelhard et al, Proc. Nat. Acad. Sci. 91, 3224-3227, 1994).
  • a number of viral-based expression systems can be used to express MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides in mammalian host cells.
  • sequences encoding MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides can be ligated into an adenovirus transcription translation complex comprising the late promoter and tripartite leader sequence.
  • Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus that is capable of expressing a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. 81, 3655-3659, 1984).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • HACs Human artificial chromosomes
  • 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).
  • Specific initiation signals also can be used to achieve more efficient translation of sequences encoding MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert.
  • Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic.
  • the efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al, Results Probl. Cell Differ. 20, 125-162, 1994).
  • a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function.
  • Different host cells that have specific cellular machinery and characteristic mechanisms for post-translational activities e.g., CHO, HeLa, MDCK, HEK293, and WI38
  • ATCC American Type Culture Collection
  • Stable expression is preferred for long-term, high-yield production of recombinant proteins.
  • cell lines which stably express MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced MuT/nudix nucleoside triphosphate pyrophosphohydrolase sequences.
  • Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, ANIMAL CELL CULTURE, R.I. Freshney, ed., 1986.
  • herpes simplex virus thymidine kinase (Wigler et al, Cell 11, 223-32, 1977) and adenine phosphoribosyltransferase (Lowy et al, Cell 22, 817-23, 1980) genes which can be employed in tk ⁇ or aprf cells, respectively.
  • antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to mefhotrexate (Wigler et al, Proc. Natl. Acad. Sci.
  • npt confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al, J. Mol. Biol. 150, 1-14, 1981), and als axi ⁇ pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murray, 1992, supra). Additional selectable genes have been described. For example, trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Nat Acad. Sci. 85, 8047-51, 1988).
  • Visible markers such as anthocyanins, ⁇ -glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al, Methods Mol. Biol. 55, 121-131, 1995).
  • MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide is inserted within a marker gene sequence, transformed cells containing sequences that encode a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotide.
  • host cells which contain a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotide and which express a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques that include membrane, solution, or chip-based technolo- gies for the detection and/or quantification of nucleic acid or protein.
  • the presence of a polynucleotide sequence encoding a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding a MuT/nudix nucleoside triphosphate pyrophosphohy- drolase polypeptide.
  • Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide to detect transformants that contain a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotide.
  • a variety of protocols for detecting and measuring the expression of a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal- based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can be used, or a competitive binding assay can be employed.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • sequences encoding a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can be cloned into a vector for the production of an mRNA probe.
  • RNA probes are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the polypeptide produced by a transformed cell can be secreted or contained intra- cellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides can be designed to contain signal sequences which direct secretion of soluble MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane- bound MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide.
  • MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purifica- tion on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.).
  • cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the MuT/nudix nucleoside tri- phosphate pyrophosphohydrolase polypeptide also can be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by LMAC (immobilized metal ion affinity chromatography, as described in Porath et al, Prot. Exp. Purifi 3, 263-281,
  • enterokinase cleavage site provides a means for purifying the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide from the fusion protein.
  • Vectors that contain fusion proteins are disclosed in Kroll et al, DNA Cell Biol. 12, 441-453, 1993.
  • Sequences encoding a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al, Nucl. Acids Res. Symp. Ser. 215-223, 1980;
  • MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrif ⁇ eld, J Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al, Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer).
  • fragments of MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • the newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N.Y., 1983).
  • the composition of a synthetic MuT/nudix nucleoside triphosphate pyrophosphohy- drolase polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the
  • any portion of the amino acid sequence of the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.
  • MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life that is longer than that of a transcript generated from the naturally occurring sequence.
  • nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences.
  • site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • Antibody as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab') 2 , and Fv, which are capable of binding an epitope of a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide.
  • Fab fragment antigen binding protein
  • F(ab') 2 fragment antigen binding protein
  • Fv fragment antigen binding protein
  • at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope.
  • epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.
  • An antibody which specifically binds to an epitope of a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • immunochemical assays such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody that specifically binds to the immunogen.
  • an antibody which specifically binds to a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay.
  • antibodies which specifically bind to MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide from solution.
  • Human MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response.
  • a carrier protein such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • various adjuvants can be used to increase the immunological response.
  • Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).
  • mineral gels e.g., aluminum hydroxide
  • surface active substances e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are especially useful.
  • Monoclonal antibodies that specifically bind to a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human
  • Monoclonal and other antibodies also can be "humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically.
  • Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions.
  • humanized antibodies can be produced using recombinant methods, as described in GB 2 188 638B.
  • Antibodies that specifically bind to a MuT/nudix nucleoside tri- phosphate pyrophosphohydrolase polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.
  • single chain antibodies can be adapted using methods known in the art to produce single chain antibodies that specifically bind to MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides.
  • Antibodies with related specificity, but of distinct idiotypic composition can be generated by chain shuffling from random combinatorial immuno- globin libraries (Burton, Proc. Natl. Acad. Sci. 88, 11120-23, 1991).
  • Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al, 1996, Ewr. J Cancer Prev. 5, 507-11).
  • Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in
  • a nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below.
  • single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et al, 1995, Int. J. Cancer 61, 497-501; Nicholls et al, 1993, J. Immunol. Meth. 165, 81-91).
  • Antibodies which specifically bind to MuT/nudix nucleoside triphosphate pyrophos- phohydrolase polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al, Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter et al, Nature 349, 293-299, 1991).
  • chimeric antibodies can be constructed as disclosed in WO 93/03151.
  • Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94/13804, also can be prepared.
  • Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
  • Antisense oligonucleotides are nucleotide sequences that are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of MuT/nudix nucleoside triphosphate pyrophosphohydrolase gene products in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymefhyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al,
  • Modifications of MuT/nudix nucleoside triphosphate pyrophosphohydrolase gene expression can be obtained by designing antisense oligonucleotides that will form duplexes to the control, 5', or regulatory regions of the MuT/nudix nucleoside triphosphate pyrophosphohydrolase gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons.
  • An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotide.
  • Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent MuT/nudix nucleoside triphosphate pyrophosphohydrolase nucleotides, can provide sufficient targeting specificity for MuT/nudix nucleoside triphosphate pyrophosphohydrolase mRNA.
  • each stretch of complementary contiguous nucleotides is at least
  • Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligo- nucleotide and a particular MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotide sequence.
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a MuT/nudix nucleoside triphosphate pyrophosphohydrolase poly- nucleotide. These modifications can be internal or at one or both ends of the anti- sense molecule.
  • internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
  • modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al, Trends Biotechnol 10, 152-158, 1992; Uhlmann et al, Chem. Rev. 90, 543-584, 1990; Uhlmann et al, Tetrahedron. Lett. 215, 3539-3542, 1987.
  • Ribozymes such as Agrawal et al, Trends Biotechnol 10, 152-158, 1992; Uhlmann et al, Chem. Rev. 90, 543-584, 1990; Uhlmann et al, Tetrahedron. Let
  • Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515,
  • Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al., U.S. Patent 5,641,673).
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the coding sequence of a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotide can be used to generate ribozymes that will specifically bind to mRNA transcribed from the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotide.
  • Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988).
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al, EP 321,201).
  • Specific ribozyme cleavage sites within a MuT/nudix nucleoside triphosphate pyrophosphohydrolase RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate MuT/nudix nucleoside triphosphate pyrophosphohydrolase RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a rib ⁇ zyme-containing DNA construct into cells in which it is desired to decrease MuT/nudix nucleoside triphosphate pyrophosphohydrolase expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.
  • a ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.
  • ribozymes can be engineered so that ribozyme expression will occur in response to factors that induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.
  • genes whose products interact with human MuT/nudix nucleoside triphosphate pyrophosphohydrolase may represent genes that are differentially expressed in disorders including, but not limited to, cancer, cardiovascular disorders, and CNS disorders. Further, such genes may represent genes that are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human MuT/nudix nucleoside triphosphate pyrophosphohydrolase gene or gene product may itself be tested for differential expression.
  • the degree to which expression differs in a normal versus a diseased state need only be large enough to be visualized via standard characterization techniques such as differential display techniques.
  • standard characterization techniques such as differential display techniques.
  • Other such standard characterization techniques by which expression differences may be visualized include but are not limited to, quantitative RT (reverse transcriptase), PCR, and Northern analysis.
  • RNA or, preferably, mRNA is isolated from tissues of interest.
  • RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects.
  • RNA isolation technique that does not select against the isolation of mRNA may be utilized for the purification of such RNA samples. See, for example, Ausubel et al, ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. New York, 1987-1993. Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, U.S. Patent 4,843,155.
  • Transcripts within the collected RNA samples that represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al, Proc.
  • the differential expression information may itself suggest relevant methods for the treatment of disorders involving the human MuT/nudix nucleoside triphosphate pyrophosphohydrolase.
  • treatment may include a modulation of expression of the differentially expressed genes and/or the gene encoding the human MuT/nudix nucleoside triphosphate pyrophosphohydrolase.
  • the differential expression infor- mation may indicate whether the expression or activity of the differentially expressed gene or gene product or the human MuT/nudix nucleoside triphosphate pyrophosphohydrolase gene or gene product are up-regulated or down-regulated.
  • the invention provides assays for screening test compounds that bind to or modulate the activity of a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide or a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotide.
  • a test compound preferably binds to a MuT/nudix nucleoside triphos- phate pyrophosphohydrolase polypeptide or polynucleotide. More preferably, a test compound decreases or increases MuT/nudix nucleoside triphosphate pyrophosphohydrolase activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.
  • Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity.
  • the compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, 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.
  • the biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-pep tide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.
  • Test compounds can be screened for the ability to ' bind to MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides or polynucleotides or to affect MuT/nudix nucleoside triphosphate pyrophosphohydrolase activity or MuT/nudix nucleoside triphosphate pyrophosphohydrolase gene expression using high throughput screening.
  • high throughput screening many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened.
  • microtiter plates typically require assay volumes that range from 50 to 500 ⁇ l.
  • assay volumes typically range from 50 to 500 ⁇ l.
  • many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.
  • free format assays or assays that have no physical barrier between samples, can be used.
  • an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al, Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994).
  • the cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose.
  • the combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.
  • Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UN-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.
  • test samples are placed in a porous matrix.
  • One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • the test compound is preferably a small molecule that binds to and occupies, for example, the active site of the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide, such that normal biological activity is prevented.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules.
  • either the test compound or the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • a detectable label such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • Detection of a test compound that is bound to the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.
  • binding of a test compound to a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can be determined without labeling either of the interactants.
  • a microphysiometer can be used to detect binding of a test compound with a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide.
  • a microphysiometer e.g., CytosensorTM
  • CytosensorTM is an analytical instrument that measures the rate at which a cell acidifies its environment using a light- addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide (McConnell et al,
  • BIA Bimolecular Interaction Analysis
  • a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et al, Cell 72, 223-232, 1993; Madura et al, J. Biol. Chem.
  • 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.
  • polynucleotide encoding a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence that encodes an unidentified protein (“prey” or “sample” can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the "bait" and the "prey” proteins are able to interact in vivo to form an protein-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene
  • a transcriptional regulatory site responsive to the transcription factor e.g., LacZ
  • Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein that interacts with the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide.
  • either the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide (or polynucleotide) or the test compound can be bound to a solid support.
  • Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads).
  • Any method known in the art can be used to attach the enzyme polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support.
  • Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide is a fusion protein comprising a domain that allows the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide to be bound to a solid support.
  • glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
  • the test compound or the test compound and the non-adsorbed MuT/nudix nucleoside triphosphate pyrophos- phohydrolase polypeptide are then combined with the test compound or the test compound and the non-adsorbed MuT/nudix nucleoside triphosphate pyrophos- phohydrolase polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.
  • a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide or polynucleotide
  • a test compound can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS (N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of strep avidin-coated 96 well plates (Pierce Chemical).
  • antibodies which specifically bind to a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide, can be derivatized to the wells of the plate.
  • Unbound target or protein can be trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using anti- bodies which specifically bind to the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide, and SDS gel electrophoresis under non-reducing conditions.
  • Screening for test compounds which bind to a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide or polynucleotide can be used in a cell-based assay system. A MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide or polynucleotide is determined as described above.
  • Test compounds can be tested for the ability to increase or decrease the pyrophosphohydrolase activity of a human MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide.
  • MuT/nudix nucleoside triphosphate pyrophosphohydrolase activity can be measured, for example, as described in Bhatnagar, S. K., Bessman, M. J. (1988) J. Biol. Chem. 263, 8953-8957; Kamath, A. V., Yanofsky, C. (1993) Gene
  • Enzyme assays can be carried out after contacting either a purified MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide, a cell membrane preparation, or an intact cell with a test compound.
  • a test compound that decreases a pyrophosphohydrolase activity of a MuT/nudix nucleoside triphosphate pyrophos- phohydrolase polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100%) is identified as a potential therapeutic agent for decreasing MuT/nudix nucleoside triphosphate pyrophosphohydrolase activity.
  • a test compound which increases a pyrophosphohydrolase activity of a human MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for increasing human MuT/nudix nucleoside triphosphate pyrophosphohydrolase activity.
  • test compounds that increase or decrease MuT/nudix nucleoside triphosphate pyrophosphohydrolase gene expression are identified.
  • a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotide is con- tacted with a test compound, and the expression of an RNA or polypeptide product of the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotide is determined.
  • the level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound.
  • the test compound can then be identified as a modulator of expression based on this comparison.
  • test compound when expression of mRNA or polypeptide is greater in the presence of the test compound tha in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression.
  • test compound when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.
  • the level of MuT/nudix nucleoside triphosphate pyrophosphohydrolase RNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used.
  • the presence of polypeptide products of a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry.
  • polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a
  • MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide.
  • Such screening can be carried out either in a cell-free assay system or in an intact cell.
  • Any cell that expresses a MuT/nudix nucleoside triphosphate pyrophosphohy- drolase polynucleotide can be used in a cell-based assay system.
  • the MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used.
  • compositions of the inven- tion can comprise, for example, a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide, MuT/nudix nucleoside triphosphate pyrophosphohydrolase polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide, or mimetics, activators, or inhibitors of a MuT/nudix nucleoside triphos- phate pyrophosphohydrolase polypeptide activity.
  • compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • agent such as stabilizing compound
  • the compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means.
  • Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i. e. , dosage.
  • Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • a filler or binders such as lactose or starches
  • lubricants such as talc or magnesium stearate
  • stabilizers optionally, stabilizers.
  • the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds can be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers also can be used for delivery.
  • the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%>-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • compositions After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.
  • Human MuT/nudix nucleoside triphosphate pyrophosphohydrolase can be regulated to treat cancer, cardiovascular disorders, and CNS disorders.
  • the MutT/nudix family of enzymes acts, in part, as "housecleaning" enzymes that eliminate deleterious compounds, such as the mutagenic form of dGTP, 8-oxo-dGTPn (2).
  • members of this family of enzymes function as modulators of nucleoside phosphate concentration in various metabolic and signaling pathways such as hydrolysis of dATP by the MutT/nudix module to prevent the untimely initiation of apoptosis and the hydrolysis of diadenosine polyphosphates Ap(n)A (p: 2-6), which have possible roles as neurotransmitters, extracellular signaling molecules and 'alarmones' secreted by cells in response to stress.
  • Oxidative stress may explain the over expression of hMTHl, a MutT containing enzyme that prevents the misincorporation of the mutagen 8-oxo-dGTP in DNA observed in lung and kidney cancers (12, 13).
  • Human MuT/nudix nucleoside triphosphate pyrophosphohydrolase may, therefore, prove useful as a prognostic marker for tumor development.
  • Diadenosine polyphosphates act as neurotransmitters at metabotropic or ionotropic receptors, or as analogues of P2X and P2Y receptors (4).
  • Intracellular diadenosine polyphosphates can be transported to secretory granules for exocytotic release.
  • the upstream pharmacological modulation of intracellular diadenosine polyphosphate concentration may have down stream effects.
  • the ionotropic receptor for example, the ionotropic receptor
  • P4 which is present in synaptic terminals, is modulated, in part, by the extracellular diadenosine polyphosphate metabolites, adenosine and ATP.
  • diadenosine polyphosphates generally produce vasodilation via mechanisms thought to involve release of NO or prostacyclin (PGL2). They produce cardiac electrophysiological effects by altering ventricular refractoriness at submicromolar concentrations and reduce heart rate.
  • P2Y receptors mediate the action of extra- cellular nucleotides in cell-to-cell signaling (9).
  • Ap(3)A activates the P2Y(1) receptor resulting in an increase in intracellular Ca2+
  • Ap(4)A, Ap(5)A, and Ap(6)A are specific antagonists of endogenous Ca2+ coupled P2Y(1) receptors (10).
  • diadenosine polyphosphates activate predominantly a Ca(2+)-dependent K(+)-conductance (11).
  • Cancer is a disease fundamentally caused by oncogenic cellular transformation. There are several hallmarks of transformed cells that distinguish them from their normal counterparts and underlie the pathophysiology of cancer. These include uncontrolled cellular proliferation, unresponsiveness to normal death-inducing signals (immortalization), increased cellular motility and invasiveness, increased ability to recruit blood supply through induction of new blood vessel formation (angiogenesis), genetic instability, and dysregulated gene expression. Various combinations of these aberrant physiologies, along with the acquisition of drug- resistance frequently lead to an intractable disease state in which organ failure and patient death ultimately ensue.
  • Genes or gene fragments identified through genomics can readily be expressed in one or more heterologous expression systems to produce functional recombinant proteins. These proteins are characterized in vitro for their biochemical properties and then used as tools in high-throughput molecular screening programs to identify chemical modulators of their biochemical activities. Activators and/or inhibitors of target protein activity can be identified in this manner and subsequently tested in cellular and in vivo disease models for anti-cancer activity. Optimization of lead compounds with iterative testing in biological models and detailed pharmacokinetic and toxicological analyses form the basis for drug development and subsequent testing in humans.
  • Cardiovascular diseases include the following disorders of the heart and the vascular system: congestive heart failure, myocardial infarction, ischemic diseases of the heart, all kinds of atrial and ventricular arrhythmias, hypertensive vascular diseases, and peripheral vascular diseases.
  • Heart failure is defined as a pathophysiologic state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirement of the metabolizing tissue. It includes all forms of pumping failure, such as high-output and low-output, acute and chronic, right-sided or left-sided, systolic or diastolic, independent of the underlying cause.
  • MI Myocardial infarction
  • Ischemic diseases are conditions in which the coronary flow is restricted resulting in a perfusion which is inadequate to meet the myocardial requirement for oxygen.
  • This group of diseases includes stable angina, unstable angina, and asymptomatic ischemia.
  • Arrhythmias include all forms of atrial and ventricular tachyarrhythmias (atrial tachycardia, atrial flutter, atrial fibrillation, atrio-ventricular reentrant tachycardia, preexcitation syndrome, ventricular tachycardia, ventricular flutter, and ventricular fibrillation), as well as bradycardic forms of arrhythmias.
  • vascular diseases include primary as well as all kinds of secondary arterial hypertension (renal, endocrine, neurogenic, others).
  • the disclosed gene and its product may be used as drug targets for the treatment of hypertension as well as for the prevention of all complications.
  • Peripheral vascular diseases are defined as vascular diseases in which arterial and or venous flow is reduced resulting in an imbalance between blood supply and tissue oxygen demand. It includes chronic peripheral arterial occlusive disease (PAOD), acute arterial thrombosis and embolism, inflammatory vascular disorders, Raynaud's phenomenon, and venous disorders.
  • PAOD peripheral arterial occlusive disease
  • acute arterial thrombosis and embolism inflammatory vascular disorders
  • Raynaud's phenomenon Raynaud's phenomenon
  • venous disorders venous disorders.
  • Central and peripheral nervous sytem disorders also can be treated, such as primary and secondary disorders after brain injury, disorders of mood, anxiety disorders, disorders of thought and volition, disorders of sleep and wakefulness, diseases of the motor unit, such as neurogenic and myopathic disorders, neurodegenerative disorders such as Alzheimer's and Parkinson's disease, and processes of peripheral and chronic pain.
  • Pain that is associated with CNS disorders also can be treated by regulating the activity of human MuT/nudix nucleoside triphosphate pyrophosphohydrolase. Pain which can be treated includes that associated with central nervous system disorders, such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemorrhage, vascular mal- formation).
  • central nervous system disorders such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemorrhage, vascular mal- formation).
  • Non-central neuropathic pain includes that associated with post mastectomy pain, reflex sympathetic dystrophy (RSD), trigeminal neuralgiaradioculopa- thy, post-surgical pain, HIV/ AIDS related pain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy, vasculitic neuropathy secondary to connective tissue disease), paraneoplastic polyneuropathy associated, for example, with carcinoma of lung, or leukemia, or lymphoma, or carcinoma of prostate, colon or stomach, trigeminal neuralgia, cranial neuralgias, and post-herpetic neuralgia. Pain associated with cancer and cancer treatment also can be treated, as can headache pain (for example, migraine with aura, migraine without aura, and other migraine disorders), episodic and chronic tension-type headache, tension-type like headache, cluster headache, and chronic paroxysmal hemicrania.
  • headache pain for example, migraine with aura, migraine without aura, and other migraine disorders
  • episodic and chronic tension-type headache tension-type like headache,
  • This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide binding molecule
  • 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.
  • a reagent which affects MuT/nudix nucleoside triphosphate pyrophosphohydrolase activity can be administered to a human cell, either in vitro or in vivo, to reduce MuT/nudix nucleoside triphosphate pyrophosphohydrolase activity.
  • the reagent preferably binds to an expression product of a human MuT/nudix nucleoside triphosphate pyrophosphohydrolase gene. If the expression product is a protein, the reagent is preferably an antibody.
  • an antibody can be added to a preparation of stem cells that have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.
  • the reagent is delivered using a liposome.
  • the liposome is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours.
  • a liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human.
  • the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin.
  • a liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell.
  • the transfection efficiency of a liposome is about 0.5 ⁇ g of DNA per 16 nmole of liposome delivered to about 10 cells, more preferably about 1.0 ⁇ g of DNA per 16 nmole of liposome delivered to about 10 cells, and even more preferably about 2.0 ⁇ g of DNA per 16 nmol of liposome delivered to about 10 6 cells.
  • a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.
  • Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to poly- ethylene glycol.
  • a liposome comprises a compound capable of targeting the liposome to a particular cell type, such as a cell-specific ligand exposed on the outer surface of the liposome.
  • a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods that are standard in the art (see, for example, U.S. Patent 5,705,151).
  • a reagent such as an antisense oligonucleotide or ribozyme
  • from about 0.1 ⁇ g to about 10 ⁇ g of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 ⁇ g to about 5 ⁇ g of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 ⁇ g of polynucleotides is combined with about 8 nmol liposomes.
  • antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery.
  • Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol 11, 202-05 (1993); Chiou et al, GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988);
  • a therapeutically effective dose refers to that amount of active ingredient which increases or decreases MuT/nudix nucleoside triphosphate pyrophosphohydrolase activity relative to the MuT/nudix nucleoside triphosphate pyrophosphohydrolase activity which occurs in the absence of the therapeutically effective dose.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic efficacy and toxicity e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50%) of the population
  • ED 50 the dose therapeutically effective in 50% of the population
  • LD 50 the dose lethal to 50% of the population
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD 5 o/ED 50 .
  • compositions that exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED 5 0 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors that can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun,” and DEAE- or calcium phosphate-mediated transfection.
  • Effective in vivo dosages of an antibody are in the range of about 5 ⁇ g to about 50 ⁇ g/kg, about 50 ⁇ g to about 5 mg/kg, about 100 ⁇ g to about 500 ⁇ g/kg of patient body weight, and about 200 to about 250 ⁇ g/kg of patient body weight.
  • effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g of DNA.
  • the reagent is preferably an antisense oligo- nucleotide or a ribozyme.
  • Polynucleotides that express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
  • a reagent reduces expression of a MuT/nudix nucleoside triphosphate pyrophosphohydrolase gene or the activity of a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100%> relative to the absence of the reagent.
  • the effectiveness of the mechanism chosen to decrease the level of expression of a MuT/nudix nucleoside triphosphate pyrophosphohydrolase gene or the activity of a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to MuT/nudix nucleoside triphosphate pyrophosphohydrolase-specific mRNA, quantitative RT-PCR, immunologic detection of a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide, or measurement of MuT/nudix nucleoside triphosphate pyrophosphohydrolase activity.
  • any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents.
  • Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • Human MuT/nudix nucleoside triphosphate pyrophosphohydrolase also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences that encode the enzyme. For example, differences can be determined between the cDNA or genomic sequence encoding MuT/nudix nucleoside triphosphate pyrophosphohydrolase in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.
  • Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method.
  • cloned DNA segments can be employed as probes to detect specific DNA segments.
  • the sensitivity of this method is greatly enhanced when combined with PCR.
  • a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.
  • DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et ⁇ /., Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et al, Proc.
  • the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA.
  • direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.
  • Altered levels of MuT/nudix nucleoside triphosphate pyrophosphohydrolase also can be detected in various tissues.
  • Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELIS A assays.
  • the polynucleotide of SEQ ID NO: 1 is inserted into the expression vector pCEN4 and the expression vector pCEN4-MuT/nudix nucleoside trisphosphate pyrophosphohydrolase polypeptide obtained is transfected into human embryonic kidney 293 cells. From these cells extracts are obtained and ran in 0.5 ml capped polypropylene tubes in an incubation mixture of total volume of 60 ⁇ l containing 5-120 ⁇ M 8-oxo- dGTP, 5 mM MgC12, 100 mM Tris-HCl buffer (pH 6-10), and 5-20 ⁇ l of the cell extract.
  • reaction is initiated by adding the extract or ultrafiltrate, carried out at 37°C for 30-120 min, and terminated by adding 20 ⁇ l of 50 mM ⁇ aEDTA.
  • the reaction mixture is finally analyzed by HPLC. To determine the reaction time course, the volume of reaction mixture is scaled up to
  • ID NO: 2 has a MuT/nudix nucleoside trisphosphate pyrophosphohydrolase activity.
  • the Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, CA) is used to produce large quantities of recombinant human MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides in yeast.
  • the MuT/nudix nucleoside triphosphate pyrophosphohydrolase-encoding DNA sequence is derived from SEQ ID NO: 1. Before insertion into vector pPICZB, the DNA sequence is modified by well known methods in such a way that it contains at its 5 '-end an initiation codon and at its 3 '-end an enterokinase cleavage site, a His6 reporter tag and a termination codon.
  • the yeast is cultivated under usual conditions in 5 liter shake flasks and the recombinantly produced protein isolated from the culture by affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea.
  • the bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invitrogen, San Diego, CA) according to manufacturer's instructions. Purified human MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide is obtained.
  • Purified MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides comprising a glutathione-S-transferase protein and absorbed onto glutathione- derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution.
  • Human MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptides comprise the amino acid sequence shown in SEQ ID NO: 2.
  • the test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.
  • the buffer solution containing the test compounds is washed from the wells.
  • Binding of a test compound to a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide is detected by fluorescence measurements of the contents of the wells.
  • a test compound that increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to a MuT/nudix nucleoside triphosphate pyrophosphohydrolase polypeptide.
  • test compound is administered to a culture of human cells transfected with a
  • MuT/nudix nucleoside triphosphate pyrophosphohydrolase expression construct and incubated at 37°C for 10 to 45 minutes. A culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control.
  • RNA is isolated from the two cultures as described in Chirgwin et al, Biochem. 18, 5294-99, 1979).
  • Northern blots are prepared using 20 to 30 ⁇ g total RNA and hybridized with a P-labeled MuT/nudix nucleoside triphosphate pyrophosphohydrolase-specific probe at 65°C in Express-hyb (CLONTECH).
  • the probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ID NO: 1.
  • a test compound that decreases the MuT/nudix nucleoside triphosphate pyrophosphohydrolase-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of MuT/nudix nucleoside triphosphate pyrophosphohydrolase gene expression.
  • a test compound is administered to a culture of human cells transfected with a MuT/nudix nucleoside triphosphate pyrophosphohydrolase expression construct and incubated at 37°C for 10 to 45 minutes.
  • a culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control.
  • MuT/nudix nucleoside triphosphate pyrophosphohydrolase activity is measured using a method of Bhatnagar, S. K., Bessman, M. J. (1988) J. Biol. Chem. 263, 8953-8957; Kama h, A. V., Yanofsky, C. (1993) Gene
  • a test compound which decreases the MuT/nudix nucleoside triphosphate pyrophosphohydrolase activity of the MuT/nudix nucleoside triphosphate pyrophosphohydrolase relative to the MuT/nudix nucleoside triphosphate pyrophosphohydrolase activity in the absence of the test compound is identified as an inhibitor of
  • MuT/nudix nucleoside triphosphate pyrophosphohydrolase activity MuT/nudix nucleoside triphosphate pyrophosphohydrolase activity.
  • MuT/nudix nucleoside triphosphate pyrophosphohydrolase The qualitative expression pattern of MuT/nudix nucleoside triphosphate pyrophosphohydrolase in various tissues is determined by Reverse Transcription- Polymerase Chain Reaction (RT-PCR).
  • RT-PCR Reverse Transcription- Polymerase Chain Reaction
  • expression is determined in the following tissues: adrenal gland, bone marrow, brain, cerebellum, colon, fetal brain, fetal liver, heart, kidney, liver, lung, mammary gland, pancreas, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, uterus, and peripheral blood lymphocytes.
  • Expression in the following cancer cell lines also is determined: DU-145 (prostate), NCI-H125 (lung), HT-29 (colon), COLO-205 (colon), A-549 (lung), NCI-H460 (lung), HT-116 (colon), DLD-1 (colon), MDA-MD-231 (breast), LS174T (colon), ZF-75 (breast), MDA-MN-435 (breast), HT-1080, MCF-7 (breast), and U87. Matched pairs of malignant and normal tissue from the same patient also are tested.
  • MuT/nudix nucleoside triphosphate pyrophosphohydrolase is involved in CNS disorders
  • the following tissues are screened: fetal and adult brain, muscle, heart, lung, kidney, liver, thymus, testis, colon, placenta, trachea, pancreas, kidney, gastric mucosa, colon, liver, cerebellum, skin, cortex (Alzheimer's and normal), hypothalamus, cortex, amygdala, cerebellum, hippocampus, choroid, plexus, thalamus, and spinal cord.
  • Quantitative expression profiling is performed by the form of quantitative PCR analysis called "kinetic analysis” firstly described in Higuchi et al, BioTechnology 10, 413-17, 1992, and Higuchi et al, BioTechnology
  • the probe is cleaved by the 5 '-3' endonuclease activity of Taq DNA polymerase and a fluorescent dye released in the medium (Holland et al, Proc. Natl. Acad. Sci. U.S.A. 88, 7276-80, 1991). Because the fluorescence emission will increase in direct proportion to the amount of the specific amplified product, the exponential growth phase of PCR product can be detected and used to determine the initial template concentration (Heid et al, Genome Res. 6, 986-94, 1996, and Gibson et al, Genome Res. 6, 995-1001, 1996).
  • the amplification of an endogenous control can be performed to standardize the amount of sample RNA added to a reaction.
  • the control of choice is the 18S ribosomal RNA. Because reporter dyes with differing emission spectra are available, the target and the endogenous control can be independently quantified in the same tube if probes labeled with different dyes are used.
  • RNA extraction and cDNA preparation Total RNA from the tissues listed above are used for expression quantification. RNAs labeled “from autopsy” were extracted from autoptic tissues with the TRIzol reagent (Life Technologies, MD) according to the manufacturer's protocol.
  • RNA Fifty ⁇ g of each RNA were treated with DNase I for 1 hour at 37°C in the following reaction mix: 0.2 U/ ⁇ l RNase-free DNase I (Roche Diagnostics, Germany); 0.4 U/ ⁇ l RNase inhibitor (PE Applied Biosystems, CA); 10 mM Tris-HCl pH 7.9; 10 mM MgCl 2 ; 50 mM NaCl; and 1 mM DTT.
  • RNA is extracted once with 1 volume of phenokchloroform:- isoamyl alcohol (24:24:1) and once with chloroform, and precipitated with 1/10 volume of 3 M NaAcetate, pH5.2, and 2 volumes of ethanol.
  • RNA from the autoptic tissues Fifty ⁇ g of each RNA from the autoptic tissues are DNase treated with the DNA-free kit purchased from Ambion (Ambion, TX). After resuspension and spectro- photometric quantification, each sample is reverse transcribed with the TaqMan Reverse Transcription Reagents (PE Applied Biosystems, CA) according to the manufacturer's protocol. The final concentration of RNA in the reaction mix is
  • Reverse transcription is carried out with 2.5 ⁇ M of random hexamer primers.
  • TaqMan quantitative analysis Specific primers and probe are designed according to the recommendations of PE Applied Biosystems; the probe can be labeled at the 5' end FAM (6-carboxy-fluorescein) and at the 3' end with TAMRA (6-carboxy- tetramethyl-rhodamine). Quantification experiments are performed on 10 ng of reverse transcribed RNA from each sample. Each determination is done in triplicate. Total cDNA content is normalized with the simultaneous quantification (multiplex PCR) of the 18S ribosomal RNA using the Pre-Developed TaqMan Assay Reagents (PDAR) Control Kit (PE Applied Biosystems, CA).
  • PDAR Pre-Developed TaqMan Assay Reagents
  • the assay reaction mix is as follows: IX final TaqMan Universal PCR Master Mix
  • the experiment is performed on an ABI Prism 7700 Sequence Detector (PE Applied Biosystems, CA).
  • fluorescence data acquired during PCR are processed as described in the ABI Prism 7700 user's manual in order to achieve better background subtraction as well as signal linearity with the starting target quantity.
  • the cell line used for testing is the human colon cancer cell line HCT116.
  • Cells are cultured in RPMI-1640 with 10-15% fetal calf serum at a concentration of 10,000 cells per milliliter in a volume of 0.5 ml and kept at 37°C in a 95%> air/5%CO atmosphere.
  • Phosphorothioate oligoribonucleotides are synthesized on an Applied Biosystems
  • oligonucleotides are ethanol-precipitated twice, dried, and suspended in phosphate buffered saline at the desired concentration. Purity of the oligonucleotides is tested by capillary gel electrophoresis and ion exchange HPLC. The purified oligonucleotides are added to the culture medium at a concentration of 10 ⁇ M once per day for seven days.
  • test oligonucleotide for seven days results in significantly reduced expression of human MuT/nudix nucleoside triphosphate pyrophosphohydrolase as determined by Western blotting. This effect is not observed with the control oligonucleotide.
  • the number of cells in the cultures is counted using an automatic cell counter. The number of cells in cultures treated with the test oligonucleotide (expressed as 100%) is compared with the number of cells in cultures treated with the control oligonucleotide.
  • the number of cells in cultures treated with the test oligonucleotide is not more than 30%> of control, indicating that the inhibition of human MuT/nudix nucleoside triphosphate pyrophosphohydrolase has an anti- proliferative effect on cancer cells.
  • This non-tumor assay measures the ability of a compound to reduce either the endogenous level of a circulating hormone or the level of hormone produced in response to a biologic stimulus.
  • Rodents are administered test compound (p.o., i.p., i.v., i.m., or s.c).
  • test compound p.o., i.p., i.v., i.m., or s.c
  • Plasma is assayed for levels of the hormone of interest. If the normal circulating levels of the hormone are too low and/or variable to provide consistent results, the level of the hormone may be elevated by a pre-treatment with a biologic stimulus (i.e., LHRH may be injected i.m. into mice at a dosage of a biologic stimulus (i.e., LHRH may be injected i.m. into mice at a dosage of a biologic stimulus (i.e., LHRH may be injected i.m. into mice at a dosage of
  • Hollow fibers are prepared with desired cell line(s) and implanted intraperitoneally and/or subcutaneously in rodents. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested in accordance with specific readout assay protocol, these may include assays for gene expression (bDNA, PCR, or Taqman), or a specific biochemical activity (i.e., cAMP levels. Results are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p ⁇ 0.05 as compared to the vehicle control group.
  • specific readout assay protocol these may include assays for gene expression (bDNA, PCR, or Taqman), or a specific biochemical activity (i.e., cAMP levels. Results are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p ⁇
  • Rodents are administered test compound (p.o., i.p., i.v., i.m., or s.c.) according to a predetermined schedule and for a predetermined duration (i.e., 1 week).
  • animals are weighed, the target organ is excised, any fluid is expressed, and the weight of the organ is recorded.
  • Blood plasma may also be collected. Plasma may be assayed for levels of a hormone of interest or for levels of test agent.
  • Organ weights may be directly compared or they may be normalized for the body weight of the animal. Compound effects are compared to a vehicle-treated control group. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test. Significance is p value ⁇ 0.05 compared to the vehicle control group.
  • Hollow fibers are prepared with desired cell line(s) and implanted mtraperitoneally and/or subcutaneously in rodents. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested in accordance with specific readout assay protocol.
  • Cell proliferation is determined by measuring a marker of cell number (i.e., MTT or LDH). The cell number and change in cell number from the starting inoculum are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p ⁇ 0.05 as compared to the vehicle control group.
  • Hydron pellets with or without growth factors or cells are implanted into a micro- pocket surgically created in the rodent cornea.
  • Compound administration may be systemic or local (compound mixed with growth factors in the hydron pellet).
  • Corneas are harvested at 7 days post implantation immediately following intracardiac infusion of colloidal carbon and are fixed in 10% > formalin. Readout is qualitative scoring and or image analysis. Qualitative scores are compared by Rank Sum test. Image analysis data is evaluated by measuring the area of neovascularization (in pixels) and group averages are compared by Student's t-test (2 tail). Significance is p ⁇ 0.05 as compared to the growth factor or cells only group.
  • Matrigel containing cells or growth factors, is injected subcutaneously. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Matrigel plugs are harvested at predetermined time point(s) and prepared for readout. Readout is an ELISA-based assay for hemoglobin concentration and/or histological examination (i.e. vessel count, special staining for endothelial surface markers: CD31, factor-8). Readouts are analyzed by Student's t-test, after the variance between groups is compared by an F- test, with significance determined at p ⁇ 0.05 as compared to the vehicle control group.
  • Tumor cells or fragments are implanted subcutaneously on Day 0.
  • Vehicle and/or compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule starting at a time, usually on Day 1, prior to the ability to measure the tumor burden.
  • Body weights and tumor measurements are recorded 2-3 times weekly. Mean net body and tumor weights are calculated for each data collection day.
  • Anti- tumor efficacy may be initially determined by comparing the size of treated (T) and control (C) tumors on a given day by a Student's t-test, after the variance between groups is compared by an F-test, with significance determined at p ⁇ 0.05.
  • Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan- Meier curves from the times for individual tumors to attain the evaluation size. Significance is p ⁇ 0.05.
  • Tumor cells are injected mtraperitoneally or intracranially on Day 0.
  • Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule starting on Day 1. Observations of morbidity and/or mortality are recorded twice daily. Body weights are measured and recorded twice weekly. Morbidity/mortality data is expressed in terms of the median time of survival and the number of long- term survivors is indicated separately. Survival times are used to generate Kaplan- Meier curves. Significance is p ⁇ 0.05 by a log-rank test compared to the control group in the experiment.
  • Tumor cells or fragments are implanted subcutaneously and grown to the desired size for treatment to begin. Once at the predetermined size range, mice are randomized into treatment groups. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Tumor and body weights are measured and recorded 2-3 times weekly. Mean tumor weights of all groups over days post inoculation are graphed for comparison. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group.
  • Tumor measurements may be recorded after dosing has stopped to monitor tumor growth delay.
  • Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p value ⁇ 0.05 compared to the vehicle control group.
  • Tumor cells or fragments, of mammary adenocarcinoma origin are implanted directly into a surgically exposed and reflected mammary fat pad in rodents. The fat pad is placed back in its original position and the surgical site is closed. Hormones may also be administered to the rodents to support the growth of the tumors. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Tumor and body weights are measured and recorded 2-3 times weekly. Mean tumor weights of all groups over days post inoculation are graphed for comparison. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group.
  • Tumor measurements may be recorded after dosing has stopped to monitor tumor growth delay.
  • Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p value ⁇ 0.05 compared to the vehicle control group.
  • Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ, or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment. 3.3.2. Intraprostatic Assay
  • Tumor cells or fragments, of prostatic adenocarcinoma origin are implanted directly into a surgically exposed dorsal lobe of the prostate in rodents.
  • the prostate is externalized through an abdominal incision so that the tumor can be implanted specifically in the dorsal lobe while verifying that the implant does not enter the seminal vesicles.
  • the successfully inoculated prostate is replaced in the abdomen and the incisions through the abdomen and skin are closed.
  • Hormones may also be administered to the rodents to support the growth of the tumors.
  • Qompounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule.
  • Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected.
  • the size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope.
  • An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group. This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor.
  • Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the lungs), or measuring the target organ weight (i.e., the regional lymph nodes). The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment.
  • Tumor cells of pulmonary origin may be implanted intrabronchially by making an incision through the skin and exposing the trachea.
  • the trachea is pierced with the beveled end of a 25 gauge needle and the tumor cells are inoculated into the main bronchus using a flat-ended 27 gauge needle with a 90° bend.
  • Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected.
  • the size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope.
  • An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group.
  • This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the contralateral lung), or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment.
  • Tumor cells of gastrointestinal origin may be implanted intracecally by making an abdominal incision through the skin and externalizing the intestine. Tumor cells are inoculated into the cecal wall without penetrating the lumen of the intestine using a 27 or 30 gauge needle. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected. The size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope.
  • An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t- test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group. This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the liver), or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment.
  • Tumor cells are inoculated s.c. and the tumors allowed to grow to a predetermined range for spontaneous metastasis studies to the lung or liver. These primary tumors are then excised. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule which may include the period leading up to the excision of the primary tumor to evaluate therapies directed at inhibiting the early stages of tumor metastasis. Observations of morbidity and/or mortality are recorded daily. Body weights are measured and recorded twice weekly. Potential endpoints include survival time, numbers of visible foci per target organ, or target organ weight. When survival time is used as the endpoint the other values are not determined.
  • Survival data is used to generate Kaplan-Meier curves. Significance is p ⁇ 0.05 by a log-rank test compared to the control group in the experiment. The mean number of visible tumor foci, as determined under a dissecting microscope, and the mean target organ weights are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment for both of these endpoints. ,
  • Tumor cells are injected into the tail vein, portal vein, or the left ventricle of the heart in experimental (forced) lung, liver, and bone metastasis studies, respectively.
  • Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Observations of morbidity and/or mortality are recorded daily. Body weights are measured and recorded twice weekly. Potential endpoints include survival time, numbers of visible foci per target organ, or target organ weight. When survival time is used as the endpoint the other values are not determined. Survival data is used to generate Kaplan-Meier curves. Significance is p ⁇ 0.05 by a log-rank test compared to the control group in the experiment.
  • the mean number of visible tumor foci, as determined under a dissecting microscope, and the mean target organ weights are compared by Student's t-test after conducting an F-test, with significance at p ⁇ 0.05 compared to the vehicle control group in the experiment for both endpoints.
  • Acute pain is measured on a hot plate mainly in rats.
  • Two variants of hot plate testing are used: In the classical variant animals are put on a hot surface (52 to 56°C) and the latency time is measured until the animals show nocifensive behavior, such as stepping or foot licking.
  • the other variant is an increasing temperature hot plate where the experimental animals are put on a surface of neutral temperature. Subsequently this surface is slowly but constantly heated until the animals begin to lick a hind paw. The temperature which is reached when hind paw licking begins is a measure for pain threshold.
  • Compounds are tested against a vehicle treated control group. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.i, Lev., s.c, intradermal, transdermal) prior to pain testing. Persistent Pain
  • Persistent pain is measured with the formalin or capsaicin test, mainly in rats.
  • a solution of 1 to 5% formalin or 10 to 100 ⁇ g capsaicin is injected into one hind paw of the experimental animal.
  • the animals show nocifensive reactions like flinching, licking and biting of the affected paw.
  • the number of nocifensive reactions within a time frame of up to 90 minutes is a measure for intensity of pain.
  • Compounds are tested against a vehicle treated control group. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., Lev., s.c, intradermal, transdermal) prior to formalin or capsaicin administration.
  • application routes i.v., i.p., p.o., i.t., Lev., s.c, intradermal, transdermal
  • Neuropathic pain is induced by different variants of unilateral sciatic nerve injury mainly in rats.
  • the operation is performed under anesthesia.
  • the first variant of sciatic nerve injury is produced by placing loosely constrictive ligatures around the common sciatic nerve.
  • the second variant is the tight ligation of about the half of the diameter of the common sciatic nerve.
  • a group of models is used in which tight ligations or transections are made of either the L5 and L6 spinal nerves, or the L%> spinal nerve only.
  • the fourth variant involves an axotomy of two of the three terminal branches of the sciatic nerve (tibial and common peroneal nerves) leaving the remaining sural nerve intact whereas the last variant comprises the axotomy of only the tibial branch leaving the sural and common nerves uninjured. Control animals are treated with a sham operation.
  • the nerve injured animals develop a chronic mechanical allodynia, cold allodynioa, as well as a thermal hyperalgesia.
  • Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, UTC Inc. -Life Science Instruments, Woodland Hills, SA, USA; Electronic von Frey System, Somedic Sales AB, H ⁇ rby, Sweden).
  • Thermal hyperalgesia is measured by means of a radiant heat source (Plantar Test, Ugo Basile, Comerio, Italy), or by means of a cold plate of 5 to 10 °C where the nocifensive reactions of the affected hind paw are counted as a measure of pain intensity.
  • a further test for cold induced pain is the counting of nocifensive reactions, or duration of nocifensive responses after plantar administration of acetone to the affected hind limb.
  • Chronic pain in general is assessed by registering the circadanian rhythms in activity (Surjo and Arndt,
  • Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t, Lev., s.c, intradermal, transdermal) prior to pain testing.
  • application routes i.v., i.p., p.o., i.t, Lev., s.c, intradermal, transdermal
  • Inflammatory pain is induced mainly in rats by injection of 0.75 mg carrageenan or complete Freund's adjuvant into one hind paw.
  • the animals develop an edema with mechanical allodynia as well as thermal hyperalgesia.
  • Mechanical allodynia is measured by means of a pressure transducer
  • Compounds are tested against uninflamed as well as vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t, Lev., s.c, intradermal, transdermal) prior to pain testing.
  • application routes i.v., i.p., p.o., i.t, Lev., s.c, intradermal, transdermal
  • Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, ETC Inc. -Life Science Instruments, Woodland Hills, S A, USA).
  • Compounds are tested against diabetic and non-diabetic vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., Lev., s.c, intradermal, transdermal) prior to pain testing.
  • application routes i.v., i.p., p.o., i.t., Lev., s.c, intradermal, transdermal
  • Degeneration of the dopaminergic nigrostriatal and striatopallidal pathways is the central pathological event in Parkinson's disease.
  • This disorder has been mimicked experimentally in rats using single/sequential unilateral stereotaxic injections of 6-OH-DA into the medium forebrain bundle (MFB).
  • Male Wistar rats (Harlan Winkelmann, Germany), weighing 200 ⁇ 250 g at the beginning of the experiment, are used.
  • the rats are maintained in a temperature- and humidity-controlled environment under a 12 h light/dark cycle with free access to food and water when not in experimental sessions.
  • the following in vivo protocols are approved by the governmental authorities.
  • Forelimb akinesia is assessed three weeks following lesion placement using a modified stepping test protocol, hi brief, the animals are held by the experimenter with one hand fixing the hindlimbs and slightly raising the hind part above the surface. One paw is touching the table, and is then moved slowly sideways (5 s for 1 m), first in the forehand and then in the backhand direction. The number of adjusting steps is counted for both paws in the backhand and forehand direction of movement. The sequence of testing is right paw forehand and backhand adjusting stepping, followed by left paw forehand and backhand directions. The test is repeated three times on three consecutive days, after an initial training period of three days prior to the first testing. Forehand adjusted stepping reveals no consistent differences between lesioned and healthy control animals. Analysis is therefore restricted to backhand adjusted stepping.
  • Balance adjustments following postural challenge are also measured during the stepping test sessions.
  • the rats are held in the same position as described in the stepping test and, instead of being moved sideways, tilted by the experimenter towards the side of the paw touching the table. This maneuver results in loss of balance and the ability of the rats to regain balance by forelimb movements is scored on a scale ranging from 0 to 3. Score 0 is given for a normal forelimb placement. When the forelimb movement is delayed but recovery of postural balance detected, score 1 is given. Score 2 represents a clear, yet insufficient, forelimb reaction, as evidenced by muscle contraction, but lack of success in recovering balance, and score 3 is given for no reaction of movement. The test is repeated three times a day on each side for three consecutive days after an initial training period of three days prior to the first testing.
  • a modified version of the staircase test is used for evaluation of paw reaching behavior three weeks following primary and secondary lesion placement.
  • Plexiglass test boxes with a central platform and a removable staircase on each side are used.
  • the apparatus is designed such that only the paw on the same side at each staircase can be used, thus providing a measure of independent forelimb use.
  • the double staircase is filled with 7 x 3 chow pellets (Precision food pellets, formula: P, purified rodent diet, size 45 mg; Sandown Scientific) on each side.
  • pellets Precision food pellets, formula: P, purified rodent diet, size 45 mg; Sandown Scientific
  • MPTP The neuro toxin l-methyl-4-phenyl-l, 2,3, 6-tetrahydro-pyri dine
  • DAergic mesencephalic dopaminergic
  • MPTP leads to a marked decrease in the levels of dopamine and its metabolites, and in the number of dopa- minergic terminals in the striatum as well as severe loss of the tyrosine hydroxylase (TH)-immunoreactive cell bodies in the substantia nigra, pars compacta.
  • TH tyrosine hydroxylase
  • mice are perfused transcardially with 0.01 M PBS (pH 7.4) for 2 min, followed by 4% parafor- maldehyde (Merck) in PBS for 15 min.
  • the brains are removed and placed in 4% paraformaldehyde for 24 h at 4°C. For dehydration they are then transferred to a 20%> sucrose (Merck) solution in 0.1 M PBS at 4°C until they sink.
  • the brains are frozen in methylbutan at -20°C for 2 min and stored at -70°C. Using a sledge microtome (mod.
  • TH free-floating tyrosine hydroxylase
  • the system logs the fall as the end of the experiment for that mouse, and the total time on the rotarod, as well as the time of the fall and all the set-up parameters, are recorded.
  • the system also allows a weak current to be passed through the base grid, to aid training.
  • the object recognition task has been designed to assess the effects of experimental manipulations on the cognitive performance of rodents.
  • a rat is placed in an open field, in which two identical objects are present.
  • the rats inspects both objects during the first trial of the object recognition task.
  • a second trial after a retention interval of for example 24 hours, one of the two objects used in the first trial, the 'familiar' object, and a novel object are placed in the open field.
  • the inspection time at each of the objects is registered.
  • the basic measures in the OR task is the time spent by a rat exploring the two object the second trial. Good retention is reflected by higher exploration times towards the novel than the 'familiar' object.
  • Administration of the putative cognition enhancer prior to the first trial predominantly allows assessment of the effects on acquisition, and eventually on consolidation processes.
  • Administration of the testing compound after the first trial allows to assess the effects on consolidation processes, whereas administration before the second trial allows to measure effects on retrieval processes.
  • the passive avoidance task assesses memory performance in rats and mice.
  • the inhibitory avoidance apparatus consists of a two-compartment box with a light compartment and a dark compartment.
  • the two compartments are separated by a guillotine door that can be operated by the experimenter.
  • a threshold of 2 cm separates the two compartments when the guillotine door is raised.
  • the illumination in the dark compartment is about 2 lux.
  • the light intensity is about 500 lux at the center of the floor of the light compartment.
  • Two habituation sessions, one shock session, and a retention session are given, separated by inter-session intervals of 24 hours, hi the habituation sessions and the retention session the rat is allowed to explore the apparatus for 300 sec.
  • the rat is placed in the light compartment, facing the wall opposite to the guillotine door. After an accommodation period of 15 sec. the guillotine door is opened so that all parts of the apparatus can be visited freely. Rats normally avoid brightly lit areas and will enter the dark compartment within a few seconds.
  • the guillotine door between the compartments is lowered as soon as the rat has entered the dark compartment with its four paws, and a scrambled 1 mA footshock is administered for 2 sec.
  • the rat is removed from the apparatus and put back into its home cage.
  • the procedure during the retention session is identical to that of the habituation sessions.
  • the step-through latency that is the first latency of entering the dark compartment (in sec.) during the retention session is an index of the memory performance of the animal; the longer the latency to enter the dark compartment, the better the retention is.
  • the Morris water escape task measures spatial orientation learning in rodents.
  • Abundant extra-maze cues are provided by the furniture in the room, including desks, computer equipment, a second water tank, the presence of the experimenter, and by a radio on a shelf that is playing softly.
  • the animals receive four trials during five daily acquisition sessions.
  • a trial is started by placing an animal into the pool, facing the wall of the tank. Each of four starting positions in the quadrants north, east, south, and west is used once in a series of four trials; their order is randomized.
  • the escape platform is always in the same position.
  • a trial is terminated as soon as the animal had climbs onto the escape platform or when 90 seconds have elapsed, whichever event occurs first. The animal is allowed to stay on the platform for 30 seconds. Then it is taken from the platform and the next trial is started. If an animal did not find the platform within 90 seconds it is put on the platform by the experimenter and is allowed to stay there for 30 seconds.
  • an additional trial is given as a probe trial: the platform is removed, and the time the animal spends in the four quadrants is measured for 30 or 60 seconds.
  • the probe trial all animals start from the same start position, opposite to the quadrant where the escape platform had been positioned during acquisition.
  • rats or mice with specific brain lesions which impair cognitive functions, or animals treated with compounds such as scopolamine or MK-801, which interfere with normal learning, or aged animals which suffer from cognitive deficits, are used.
  • the T-maze spontaneous alternation task assesses the spatial memory performance in mice.
  • the start arm and the two goal arms of the T-maze are provided with guillotine doors which can be operated manually by the experimenter.
  • a mouse is put into the start arm at the beginning of training.
  • the guillotine door is closed.
  • the 'forced trial' either the left or right goal arm is blocked by lowering the guillotine door.
  • the mouse After the mouse has been released from the start arm, it will negotiate the maze, eventually enter the open goal arm, and return to the start position, where it will be confined for 5 seconds, by lowering the guillotine door.
  • the animal can choose freely between the left and right goal arm (all guillotine-doors opened) during 14 'free choice' trials. As soon a the mouse has entered one goal arm, the other one is closed. The mouse eventually returns to the start arm and is free to visit whichever go alarm it wants after having been confined to the start arm for 5 seconds. After completion of 14 free choice trials in one session, the animal is removed from the maze. During training, the animal is never handled.
  • the percent alternations out of 14 trials is calculated. This percentage and the total time needed to complete the first forced trial and the subsequent 14 free choice trials (in s) is analyzed. Cognitive deficits are usually induced by an injection of scopolamine, 30 min before the start of the training session.
  • Scopolamine reduced the per-cent alternations to chance level, or below.
  • a cognition enhancer which is always administered before the training session, will at least partially, antagonize the scopolamine-induced reduction in the spontaneous alternation rate.
  • Diadenosine polyphosphates their biological and pharmacological significance, Bxi, et al, J Pharmacol Toxicol Methods 1995 Jun;33(3):121-8 4.
  • Diadenosine polyphosphates as antagonists of the endogenous P2Y(1) receptor in rat brain capillary endothelial cells of the B7 and B10 clones, Vigne P, Breittmayer JP, Frelin C, Br J Pharmacol 2000 Apr; 129(7): 1506-12
  • Diadenosine polyphosphates activate a Ca(2+)-dependent K(+)-conductance in porcine aortic smooth muscle cells via P2-purinoceptors, Schlatter Eet al dislike Cell Physiol Biochem 2000;10(3):125-34 2.

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Abstract

L'invention concerne des réactifs régulant la nucléoside triphosphate pyrophosphohydrolase MuT/nudix humaine et des réactifs se liant à des produits géniques de la nucléoside triphosphate pyrophosphohydrolase MuT/nudix humaine, qui peuvent jouer un rôle dans la prévention, l'amélioration ou la correction de dysfonctionnements ou de maladies, comme le cancer, les troubles cardiovasculaires et les troubles du SNC, entre autres.
PCT/EP2002/001541 2001-02-16 2002-02-14 Regulation de la nucleoside triphosphate pyrophosphohydrolase mut/nudix humaine WO2002066626A2 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000065056A2 (fr) * 1999-04-26 2000-11-02 Beth Israel Deaconess Medical Center, Inc. ACIDES NUCLEIQUES CODANT UN POLYPEPTIDE COMPRENANT UN DOMAINE $i(MUTT)

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EP1033405A3 (fr) * 1999-02-25 2001-08-01 Ceres Incorporated Fragments d'ADN avec des séquences déterminées et polypeptides encodées par lesdits fragments

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000065056A2 (fr) * 1999-04-26 2000-11-02 Beth Israel Deaconess Medical Center, Inc. ACIDES NUCLEIQUES CODANT UN POLYPEPTIDE COMPRENANT UN DOMAINE $i(MUTT)

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BESSMAN M J ET AL: "The MutT proteins or Nudix hydrolases, a family of versatile, widely distributed, housecleaning enzymes" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 271, no. 41, 11 October 1996 (1996-10-11), pages 25059-25062, XP002145837 ISSN: 0021-9258 *
DATABASE EMBL [Online] EBI, Hinxton, Cambridgeshire, U.K.; 10 March 2001 (2001-03-10) OTTENWAELDER ET AL: "Homo sapiens mRNA; cDNA DKFZp761I172 (from clone DKFZp761I172); complete cds" Database accession no. AL136592 XP002220873 *
DATABASE GENESEQ [Online] EBI, Hinxton, Cambridgeshire, U.K.; 17 October 2000 (2000-10-17) ALEXANDROV ET AL: "Arabidopsis thaliana protein fragment SEQ ID NO: 4116" Database accession no. AAG07096 XP002220876 & EP 1 033 405 A (CERES INC) 6 September 2000 (2000-09-06) *
DATABASE SWISSPROT [Online] EBI, Hinxton, Cambridgeshire, U.K.; 1 October 1996 (1996-10-01) RIEGER ET AL: "NADH pyrophosphatase (S.cerevisiae)" Database accession no. P53164 XP002220875 *
DATABASE SWISSPROT [Online] EBI, Hinxton, Cambridgesshire, U.K.; 1 June 2001 (2001-06-01) WIEMANN ET AL: "Hypothetical protein (DKFZ761l172)" Database accession no. Q9BQG2 XP002220874 *
FUJII Y ET AL: "Functional significance of the conserved residues for the 23-residue module among MTH1 and MutT family proteins." THE JOURNAL OF BIOLOGICAL CHEMISTRY. UNITED STATES 31 DEC 1999, vol. 274, no. 53, 31 December 1999 (1999-12-31), pages 38251-38259, XP002220871 ISSN: 0021-9258 *
FURUICHI M ET AL: "Genomic structure and chromosome location of the human mutT homologue gene MTH1 encoding 8-oxo-dGTPase for prevention of A:T to C:G transversion." GENOMICS. UNITED STATES DEC 1994, vol. 24, no. 3, December 1994 (1994-12), pages 485-490, XP002220870 ISSN: 0888-7543 *
GASMI L ET AL: "Cloning, expression and characterization of YSA1H, a human adenosine 5'-diphosphosugar pyrophosphatase possessing a MutT motif." THE BIOCHEMICAL JOURNAL. ENGLAND 1 DEC 1999, vol. 344 Pt 2, 1 December 1999 (1999-12-01), pages 331-337, XP002220869 ISSN: 0264-6021 *
KOONIN EUGENE V: "A highly conserved sequence motif defining the family of MuT-related proteins from eubacteria, eukaryotes and viruses" NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 21, no. 20, 1993, page 4847 XP002190555 ISSN: 0305-1048 *
MCLENNAN A G: "The MutT motif family of nucleotide phosphohydrolases in man and human pathogens (review)." INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE. GREECE JUL 1999, vol. 4, no. 1, July 1999 (1999-07), pages 79-89, XP001120292 ISSN: 1107-3756 *
MORRIS A J: "Nucleoside triphosphate pyrophosphohydrolase (NTPH)." METHODS IN ENZYMOLOGY. UNITED STATES 1978, vol. 51, 1978, pages 275-285, XP001120280 ISSN: 0076-6879 *
SAKUMI K ET AL: "CLONING AND EXPRESSION OF CDNA FOR A HUMAN ENZYME THAT HYDROLYZES 8-OXO-DGTP, A MUTAGENIC SUBSTRATE FOR DNA SYNTHESIS" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 268, no. 31, 1993, pages 23524-23530, XP000887210 ISSN: 0021-9258 *
YANG H ET AL: "CLONING AND CHARACTERIZATION OF A NEW MEMBER OF THE NUDIX HYDROLASES FROM HUMAN AND MOUSE" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 275, no. 12, 24 March 2000 (2000-03-24), pages 8844-8853, XP000938757 ISSN: 0021-9258 *

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