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WO1996015222A1 - MutT2 HUMAIN - Google Patents

MutT2 HUMAIN Download PDF

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Publication number
WO1996015222A1
WO1996015222A1 PCT/US1994/013187 US9413187W WO9615222A1 WO 1996015222 A1 WO1996015222 A1 WO 1996015222A1 US 9413187 W US9413187 W US 9413187W WO 9615222 A1 WO9615222 A1 WO 9615222A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
hmutt2
dna
polynucleotide
sequence
Prior art date
Application number
PCT/US1994/013187
Other languages
English (en)
Inventor
Ying-Fei Wei
Ewen F. Kirkness
Original Assignee
Human Genome Sciences, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Human Genome Sciences, Inc. filed Critical Human Genome Sciences, Inc.
Priority to JP8516005A priority Critical patent/JPH10509320A/ja
Priority to EP95903538A priority patent/EP0795006A4/fr
Priority to PCT/US1994/013187 priority patent/WO1996015222A1/fr
Priority to AU12558/95A priority patent/AU1255895A/en
Publication of WO1996015222A1 publication Critical patent/WO1996015222A1/fr
Priority to US08/916,989 priority patent/US6103871A/en
Priority to US09/432,253 priority patent/US6344547B1/en
Priority to US09/974,800 priority patent/US6552174B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is human MutT2, sometimes hereinafter referred to as "hMutT2. " The invention also relates to inhibiting the action of such polypeptides.
  • One type of DNA damage caused by oxygen radicals is an oxidized form of the guanine base (8-oxoguanine) (Shibutani, S., et al. , Nature, 349:431-4 (1991) ) .
  • This oxidized orm of guanine can pair with cytosine and adenine, and G:C to T:A transversions follow (T eshelashvili, L.K., et al . , J. Biol. Chem. , 266:6401-6406 (1991)).
  • active oxygen species produced by cellular metabolic intermediates are sufficient to oxidize the guanine base of the DNA, even in normally growing cells.
  • Oxidation of guanine proceeds also in a form of free nucleotide, and an oxidized form of dGTP, 8-oxo-dGTP, is a potent mutagenic substrate for DNA synthesis (Maki, H. and Sekiguchi, M., Nature, 355:273-275 (1992)).
  • 8-oxoguanine arising in DNA, 8-oxo-dGTP can induce A:T to C:G as well as G:C to T:A transversions (Cheng, K.C., et al . , J. Biol. Chem. , 267:166-172 (1992)).
  • 8-oxo-dGMP mi ⁇ incorporated opposite to dA residues of template may be removed by the mutM protein before the next round of DNA replication.
  • the mutT protein therefore, degrades the potent mutagenic substrate, 8-oxo- dGTP to the harmless monophosphate substrate to ensure proper DNA synthesis.
  • Mutations in the E. coli mutT gene cause an increase of the occurrence of A:T to C:G transversions 100- 10,000-fold over the wild-type level (Akiyama, M. , et al . , Mol. and Gen. Genet., 206:9-16 (1987)).
  • Eukaryotes and mammals also have an enzyme which hydrolyses oxidized nucleotides.
  • the enzyme is homologous to the E. coli mutT gene.
  • a significant amount of 8-oxoguanine is formed in the chromosome DNA of mammalian cells and most of the damaged nucleotides are excised from the DNA and excreted into the urine (Ames, B.N. and Gold, L.S., Mutat. Res., 250:3-16 (1991) and Shigenaga, M.K., et al . , PNAS, 86:9697-9701 (1989)) .
  • polypeptide of the present invention corresponds in size and amino acid sequence homology to human MutT and has, therefore, been preliminarily characterized as human MutT2.
  • a novel mature polypeptide which is hMutT2, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
  • isolated nucleic acid molecules encoding hMutT2 including mRNAs, DNAs, cDNAs, genomic DNA as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
  • a process for producing such polypeptide by recombinant techniques which comprises culturing recombinant prokaryotic and/or eukaryotic host cells, containing a hMutT2 nucleic acid sequence, under conditions promoting expression of said protein and subsequent recovery of said protein.
  • a process for utilizing such polypeptide, or polynucleotide encoding such polypeptide for therapeutic purposes for example, to prevent and treat diseases associated with errors in DNA replication and abnormal cell growth, for example that present in a tumor and a cancer, by specifically hydrolyzing oxidized nucleoside triphosphates, in particular, 8-oxo-dGTP, to the corresponding monophosphate for high fidelity of DNA synthesis.
  • nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to hMutT2 sequences.
  • a method of diagnosing a disease or a susceptibility to a disease for example, abnormal cellular growth, related to a mutation in hMutT2 nucleic acid sequences and the protein encoded by such nucleic acid sequences.
  • antagonists to such polypeptides which may be used to inhibit the action of such polypeptides, for example, in the treatment of tumors.
  • Figure 1 shows the cDNA sequence and the corresponding deduced amino acid sequence of hMutT2 polypeptide.
  • the standard one-letter abbreviation for amino acids are used.
  • Figure 2 illustrates the amino acid homology between E. coli MutT, human MutTl and hMutT2, wherein the shaded areas represent amino acid residues which are the same between the different sequences.
  • hMutT2 of the present invention has a higher amino acid homology to E. coli MutT than human MutTl.
  • FIG. 3 is an illustration of a gel after bacterial expression and purification of hMutT2, wherein hMutT2 is electrophoresed through the gel.
  • Lane 1 is a molecular weight marker
  • lane 2 is an E. coli. extract
  • noninduced lane 3 is an E. coli extract
  • IPTG-induced lane 4 is a nickel column purified example of lane 3.
  • Two protein bands were induced in lane 3 and 4 with molecular weight 34 kD and 31 kD with the 31 kD protein being presumably the degradation product of the 34 kD human MutT2.
  • Figure 4 is a gel showing human tissue distribution of hMutT2.
  • Lane 1 thymus; lane 2, testis; lane 3, gall bladder; lane 4, kidney,- lane 5, liver; lane 6, lung; lane 7, spleen; lane 8, prostate; lane 9, brain; lane 10, heart; lane 11, placenta.
  • hMutT2 is ubiquitously expressed in most human tissues.
  • nucleic acids which encode for the mature polypeptide having the deduced amino acid sequence of Figure 1 or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. 75882 on August 31, 1994.
  • a polynucleotide encoding a polypeptide of the present invention may be obtained from most human tissues, such as thymus, liver, spleen and prostate.
  • the polynucleotide of this invention was discovered in a cDNA library derived from a human 8 week old embryo. It is structurally related to the hMutT family. It contains an open reading frame encoding a protein of 219 amino acid residues. The protein exhibits the highest degree of homology to E.coli MutT with 62% identity and 77% similarity over a 27 amino acid stretch. It is also important that GETXE and RELQ/EEE are conserved among E.coli MutT, human MutTl and hMutT2.
  • the polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
  • the DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand.
  • the coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of Figure 1 or the deposited cDNA.
  • the polynucleotide which encodes for the mature polypeptide of Figure 1 or for the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non- coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
  • polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
  • the present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone.
  • the variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
  • the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone.
  • Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
  • the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1 or of the coding sequence of the deposited clone.
  • an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
  • the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention.
  • the marker sequence may be a hexa- histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
  • the present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 50% and preferably 70% identity between the sequences.
  • the present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • the polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNA of Figure 1 or the deposited cDNA.
  • the present invention further relates to a hMutT2 polypeptide which has the deduced amino acid sequence of Figure 1 or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide.
  • fragment, " “derivative” and “analog” when referring to the polypeptide of Figure 1 or that encoded by the deposited cDNA, means a polypeptide which retains essentially the same biological function or activity as such polypeptide.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
  • the fragment, derivative or analog of the polypeptide of Figure 1 or that encoded by the deposited cDNA may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol) , or (iv) one in which the additional amino acids are fused to the mature polypeptide.
  • a conserved or non-conserved amino acid residue preferably a conserved amino acid residue
  • substituted amino acid residue may or may not be one encoded by the genetic code
  • one or more of the amino acid residues includes a substituent group
  • the mature polypeptide is fuse
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) .
  • a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the hMutT2 genes.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques.
  • the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide.
  • Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmid ⁇ ; phage DNA; baculovirus; yeast pla ⁇ mids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenoviru ⁇ , fowl pox virus, and pseudorabies.
  • any other vector may be used as long as it is replicable and viable in the host.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence (s) (promoter) to direct mRNA synthesis.
  • s expression control sequence
  • promoters there may be mentioned: LTR or SV40 promoter, the E. coli. lac or. trp. the phage lambda P L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. Tne vector may also include appropriate sequences for amplifying expression.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • the vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
  • bacterial cells such as E. coli. Streptomyces. Salmonella typhimurium
  • fungal cells such as yeast
  • insect cells such as Drosophila S2 and Sf9
  • animal cells such as CHO, COS or Bowes melanoma,- adenoviruses,- plant cells, etc.
  • the selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • a promoter operably linked to the sequence.
  • Bacterial pQE70, pQE60, pQE-9 (Qiagen) , pBS, pDIO, phagescript, psiX174, pBluescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene) ; ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia).
  • Eukaryotic pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) .
  • any other plasmid or vector may be used as long as they are replicable and viable in the host.
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • Two appropriate vectors are pKK232-8 and pCM7.
  • Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P R , P L and trp.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the present invention relates to host cells containing the above-described constructs.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation. (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986) ) .
  • constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
  • Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al. , Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRPl gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
  • promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK) , ⁇ -factor, acid phosphatase, or heat shock proteins, among others.
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.
  • the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyce ⁇ , and Staphylococcus, although others may al ⁇ o be er ⁇ loyed a ⁇ a matter of choice.
  • useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017) .
  • cloning vector pBR322 ATCC 37017
  • Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA) .
  • pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • Cells are typically harvested by centrifugation, di ⁇ rupted by phy ⁇ ical or chemical mean ⁇ , and the re ⁇ ulting crude extract retained for further purification.
  • Microbial cells employed in expression of protein ⁇ can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.
  • mammalian cell culture sy ⁇ tem ⁇ can al ⁇ o be employed to express recombinant protein.
  • Example ⁇ of mammalian expre ⁇ sion systems include the COS-7 lines of monkey kidney fibrobla ⁇ t ⁇ , de ⁇ cribed by Gluzman, Cell, 23:175 (1981) , and other cell line ⁇ capable of expre ⁇ ing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell line ⁇ .
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor site ⁇ , transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation site ⁇ may be used to provide the required nontranscribed genetic elements.
  • the hMutT2 polypeptides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • the hMutT2 polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant technique ⁇ from a prokaryotic or eukaryotic ho ⁇ t (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) .
  • a prokaryotic or eukaryotic ho ⁇ t for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture
  • the polypeptides of the present invention may be glycosylated or may be non-glycosylated.
  • Polypeptides of the invention may also include an initial methionine amino acid residue.
  • hMutT2 polypeptides and agonists and antagonists which are polypeptides may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which i ⁇ often referred to a ⁇ "gene therapy.”
  • cell ⁇ from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
  • DNA or RNA polynucleotide
  • cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.
  • cells may be engineered in vivo for expres ⁇ ion of a polypeptide in vivo by, for example, procedure ⁇ known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the pre ⁇ ent invention may be admini ⁇ tered to a patient for engineering cell ⁇ in vivo and expression of the polypeptide in vivo.
  • the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
  • the hMutT2 polypeptide Once the hMutT2 polypeptide is being expre ⁇ ed intra- cellularly via gene therapy, it hydrolyses the oxidized form of nucleoside tripho ⁇ phates and ha ⁇ a ⁇ trong affinity for the oxidized form of guanine nucleotide, therefore eliminating them from the nucleotide pool and ensuring the high fidelity of DNA synthesi ⁇ . In the ab ⁇ ence of the hMutT2 polypeptide, there would be a significant increase in errors in DNA replication which would lead to mutagenesi ⁇ . Mutagene ⁇ i ⁇ i ⁇ known to cause numerous disorders, including abnormal cell growth, for example that present in a tumor and a cancer.
  • administration of the polypeptide of the present invention may be u ⁇ ed to treat or prevent mutagene ⁇ i ⁇ .
  • Fragments of the full length hMutT2 gene may be used as a hybridization probe for a cDNA library to isolate the full length gene and to isolate other genes which have a high sequence similarity to the gene or similar biological activity. Probes of this type can be, for example, between 20 and 2000 bases. Preferably, however, the probes have between 30 and 50 base pairs. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete hMutT2 gene including regulatory and promotor regions, exons, and intron ⁇ .
  • An example of a screen comprises isolating the coding region of the hMutT2 gene by using the known DNA sequence to synthesize an oligonucleotide probe.
  • Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • Thi ⁇ invention i ⁇ al ⁇ o related to the use of the hMutT2 gene a ⁇ part of a diagnostic as ⁇ ay for detecting diseases or susceptibility to disease ⁇ related to the presence of mutated hMutT2.
  • diseases are related to errors in DNA replication, for example, such as those which lead to tumor ⁇ and cancers.
  • Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tis ⁇ ue biopsy and autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by u ⁇ ing PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analy ⁇ is.
  • RNA or cDNA may also be used for the same purpose.
  • PCR primers complementary to the nucleic acid encoding hMutT2 can be used to identify and analyze hMutT2 mutations.
  • deletions and insertion ⁇ can be detected by a change in ⁇ ize of the amplified product in compari ⁇ on to the normal genotype.
  • Point mutation ⁇ can be identified by hybridizing amplified DNA to radiolabeled hMutT2 RNA or alternatively, radiolabeled hMutT2 anti ⁇ ense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
  • DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragment ⁇ in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high re ⁇ olution gel electrophore ⁇ i ⁇ .
  • DNA fragment ⁇ of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragment ⁇ are retarded in the gel at different po ⁇ itions according to their specific melting or partial melting temperatures (see, e.g., Myers et al . , Science, 230:1242 (1985)).
  • Sequence changes at specific locations may also be revealed by nuclease protection assays, such a ⁇ RNase and SI protection or the chemical cleavage method (e.g., Cotton et al . , PNAS, USA, 85:4397-4401 (1985)).
  • nuclease protection assays such as a ⁇ RNase and SI protection or the chemical cleavage method (e.g., Cotton et al . , PNAS, USA, 85:4397-4401 (1985)).
  • the detection of a ⁇ pecific DNA ⁇ equence may be achieved by method ⁇ ⁇ uch a ⁇ hybridization, RNa ⁇ e protection, chemical cleavage, direct DNA ⁇ equencing or the use of restriction enzymes, (e.g., Restriction Fragment Length Polymorphi ⁇ ms (RFLP) ) and Southern blotting of genomic DNA.
  • restriction enzymes e.g., Restriction Fragment Length Polymorphi ⁇ ms (RFLP)
  • mutation ⁇ can al ⁇ o be detected by in situ analy ⁇ i ⁇ .
  • the present invention also relates to a diagnostic assay for detecting altered levels of hMutT2 protein in various tissues ⁇ ince an over-expression of the proteins compared to normal control tissue samples may detect the presence of a disea ⁇ e or ⁇ u ⁇ ceptibility to a di ⁇ ea ⁇ e related to error ⁇ in DNA replication, for example, a tumor.
  • Assays used to detect levels of hMutT2 protein in a sample derived from a host are well-known to those of skill in the art and include radioimmunoassay ⁇ , competitive-binding assays, Western Blot analysis, ELISA assays and "sandwich” assay.
  • An ELISA assay (Coligan, et al., Current Protocol ⁇ in Immunology, 1(2), Chapter 6, (1991)) initially compri ⁇ es preparing an antibody specific to the hMutT2 antigen, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody.
  • a detectable reagent such as radioactivity, fluorescence or, in this example, a horseradi ⁇ h peroxida ⁇ e enzyme.
  • a sample i ⁇ removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein like BSA.
  • the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any hMutT2 proteins attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer.
  • the reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to hMutT2. Unattached reporter antibody is then washed out. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a mea ⁇ urement of the amount of hMutT2 protein present in a given volume of patient sample when compared against a standard curve.
  • a competition as ⁇ ay may be employed wherein antibodie ⁇ ⁇ pecific to hMutT2 are attached to a ⁇ olid support and labeled hMutT2 and a sample derived from the host are passed over the solid support and the amount of label detected, for example by liquid scintillation chromatography, can be correlated to a quantity of hMutT2 in the sample.
  • a "sandwich” as ⁇ ay is similar to an ELISA as ⁇ ay.
  • hMutT2 is passed over a ⁇ olid support and binds to antibody attached to a solid support.
  • a second antibody is then bound to the hMutT2.
  • a third antibody which is labeled and specific to the second antibody is then pas ⁇ ed over the solid ⁇ upport and bind ⁇ to the second antibody and an amount can then be quantitated.
  • the invention al ⁇ o relate ⁇ to a method of ⁇ creening compounds to identify those which enhance (agonists) or block (antagonists) the functions of hMutT2.
  • An example of such an assay comprises measuring the hydrolysis of 8-oxo-dGTP to 8- oxo-dGMP in the presence of hMutT2 and the compound to be screened.
  • the reaction mixture (12.5 ⁇ l) contains 20 mM Tris-HCl, pH 8.0, 4 mm MgCl 2 , 40 mM NaCl, 20 ⁇ M 8-oxo-dGTP, 80 ⁇ g/ml bovine serum albumin, 8 mM DTT, 10% glycerol, hMutT2 and the compound to be screened.
  • the reaction i ⁇ run at 30°C for 20 minute ⁇ and stopped by adding 2.0 ⁇ l of 50 mm EDTA.
  • hMutT2 Human MutT2 is produced and functions intra-cellularly, therefore, any antagonists must be intra-cellular.
  • Potential antagonists to hMutT2 include antibodies which are produced intra-cellularly.
  • an antibody identified as antagonizing hMutT2 may be produced intra-cellularly as a single chain antibody by procedures known in the art, such as transforming the appropriate cells with DNA encoding the single chain antibody to prevent the function of hMutT2.
  • Another potential hMutT2 antagonist is an anti ⁇ ense construct prepared using anti ⁇ en ⁇ e technology.
  • Anti ⁇ ense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
  • the 5' coding portion of the polynucleotide sequence which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the hMutT2 polypeptide (antisense - Okano, J. Neurochem. , 56:560 (1991); 01igodeoxynucleotides a ⁇ Antisense Inhibitors of Gene Expression, CRC Pres ⁇ , Boca Raton, FL (1988)).
  • the oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of hMutT2.
  • Potential hMutT2 antagonists also include a small molecule, which are able to pas ⁇ through the cell membrane, and bind to and occupy the catalytic ⁇ ite of the polypeptide thereby making the catalytic ⁇ ite inacce ⁇ sible to substrate such that normal biological activity is prevented.
  • small molecule ⁇ include but are not limited to ⁇ mall peptides or peptide-like molecules.
  • the antagonists may be employed to target undesired cell ⁇ , e.g., abnormally differentiating cell ⁇ such a ⁇ in tumors and cancer ⁇ , ⁇ ince the prevention of hMutT2 activity prevent corrections in DNA replication errors, and may lead to the destruction of the cell.
  • undesired cell ⁇ e.g., abnormally differentiating cell ⁇ such a ⁇ in tumors and cancer ⁇
  • ⁇ ince the prevention of hMutT2 activity prevent corrections in DNA replication errors, and may lead to the destruction of the cell.
  • the antagonist ⁇ may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
  • the small molecule agonist ⁇ and antagoni ⁇ ts of the present invention may be employed in combination with a suitable pharmaceutical carrier.
  • a suitable pharmaceutical carrier include ⁇ but i ⁇ not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • a carrier include ⁇ but i ⁇ not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the formulation should suit the mode of administration.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical composition ⁇ of the invention.
  • Associated with such container(s) can be a notice in the form pre ⁇ cribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the pharmaceutical compositions may be employed in conjunction with other therapeutic compounds.
  • compositions may be administered in a convenient manner such as by the oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route ⁇ .
  • the pharmaceutical compo ⁇ ition ⁇ are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, they are administered in an amount of at least about 10 ⁇ g/kg body weight and in most cases they will be administered in an amount not in excess of about 8 mg/Kg body weight per day. In most case ⁇ , the do ⁇ age is from about 10 ⁇ g/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.
  • sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disea ⁇ e.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
  • mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
  • ⁇ ublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner.
  • Other mapping ⁇ trategie ⁇ that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be u ⁇ ed to provide a precise chromosomal location in one step.
  • This technique can be u ⁇ ed with cDNA a ⁇ short a ⁇ 500 or 600 bases,- however, clone ⁇ larger than 2,000 bp have a higher likelihood of binding to a unique chromo ⁇ omal location with ⁇ ufficient signal intensity for simple detection.
  • FISH requires use of the clones from which the EST was derived, and the longer the better.
  • a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This a ⁇ sumes 1 megabase mapping resolution and one gene per 20 kb) .
  • the polypeptides, their fragments or other derivative ⁇ , or analogs thereof, or cells expre ⁇ ing them can be used a ⁇ an immunogen to produce antibodie ⁇ thereto.
  • the ⁇ e antibodie ⁇ can be, for example, polyclonal or monoclonal antibodie ⁇ .
  • the present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expres ⁇ ion library. Variou ⁇ procedure ⁇ known in the art may be u ⁇ ed for the production of such antibodies and fragments.
  • Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptide ⁇ . Such antibodie ⁇ can then be u ⁇ ed to i ⁇ olate the polypeptide from ti ⁇ sue expressing that polypeptide.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodie ⁇ (Cole, et al., 1985, in Monoclonal Antibodie ⁇ and Cancer Therapy, Alan R. Li ⁇ s, Inc., pp. 77-96).
  • Plasmids are designated by a lower case p preceded and/or followed by capital letters and/or numbers.
  • the starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmid ⁇ in accord with published procedure ⁇ .
  • equivalent pla ⁇ id ⁇ to those described are known in the art and will be apparent to the ordinarily skilled artisan.
  • “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA.
  • the variou ⁇ re ⁇ triction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan.
  • For analytical purposes typically 1 ⁇ g of plasmid or DNA fragment i ⁇ u ⁇ ed with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al . , Nucleic Acids Res., 8:4057 (1980).
  • Oligonucleotides refers to either a ⁇ ingle ⁇ tranded polydeoxynucleotide or two complementary polydeoxynucleotide ⁇ trand ⁇ which may be chemically ⁇ ynthe ⁇ ized. Such ⁇ ynthetic oligonucleotide ⁇ have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
  • Ligase refers ⁇ to the process of forming phosphodie ⁇ ter bonds between two double stranded nucleic acid fragment ⁇ (Maniati ⁇ , T. , et al., Id., p. 146). Unle ⁇ s otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragment ⁇ to be ligated.
  • ligase T4 DNA ligase
  • the DNA sequence encoding hMutT2, ATCC # 75882 is initially amplified using PCR oligonucleotide primers corresponding to the 5' sequence of the hMutT2 protein (minus the signal peptide sequence) and the vector sequences 3' to the hMutT2 gene. Additional nucleotides corresponding to hMutT2 were added to the 5' and 3' sequences respectively.
  • the 5' oligonucleotide primer has the sequence 5' GCGGTO ⁇ CATGAGCCAAGAACCAACG 3' contains a Sail re ⁇ triction enzyme ⁇ ite followed by 21 nucleotide ⁇ of hMutT2 coding sequence starting from the presumed terminal amino acid of the processed protein.
  • the 3' sequence 5' GCGTCTAGAT AAAATTTCAAGAAGGGCAC 3' contains complementary sequences to Xbal site and is followed by 21 nucleotides of hMutT2.
  • the restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expres ⁇ ion vector pQE-9 (Qiagen, Inc. 9259 Eton Avenue, Chat ⁇ worth, CA, 91311).
  • pQE-9 encodes antibiotic resi ⁇ tance (Amp r ) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/0) , a ribo ⁇ ome binding ⁇ ite (RBS) , a 6-Hi ⁇ tag and re ⁇ triction enzyme sites.
  • pQE-9 was then digested with Sail and Xbal.
  • the amplified sequences were ligated into pQE-9 and were inserted in frame with the sequence encoding for the histidine tag and the RBS.
  • the ligation mixture wa ⁇ then used to transform E. coli strain ml5/REP4 (Qiagen) by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Pres ⁇ ,
  • ml5/REP4 contains multiple copies of the plasmid pREP4, which expre ⁇ ses the lad repressor and al ⁇ o confer ⁇ kanamycin re ⁇ i ⁇ tance (Kan r ) .
  • Tran ⁇ formant ⁇ are identified by their ability to grow on LB plates and ampicillin/kanamycin resi ⁇ tant colonies were selected. Plasmid DNA was isolated and confirmed by re ⁇ triction analysis. Clones containing the desired construct ⁇ were grown overnight (0/N) in liquid culture in LB media ⁇ upplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) .
  • the O/N culture i ⁇ used to inoculate a large culture at a ratio of 1:100 to 1:250.
  • the cells were grown to an optical density 600 (O.D, 600 ) of between 0.4 and 0.6.
  • IPTG n Isopropyl-B-D-thiogalacto pyranoside
  • IPTG induces by inactivating the la repre ⁇ or, clearing the P/0 leading to increased gene expression.
  • Cells were grown an extra 3 to 4 hour ⁇ . Cells were then harvested by centrifugation. The cell pellet was ⁇ olubilized in the chaotropic agent 6 Molar Guanidine HC1.
  • ⁇ olubilized hMutT2 wa ⁇ purified from thi ⁇ solution by chromatography on a Nickel- Chelate column under condition ⁇ that allow for tight binding by protein ⁇ containing the 6-Hi ⁇ tag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984)) .
  • hMutT2 (more than 80% pure) wa ⁇ eluted from the column in 6 molar guanidine HC1 pH 5.0 and for the purpo ⁇ e of renaturation adju ⁇ ted to 3 molar guanidine HC1, lOOmM ⁇ odium pho ⁇ phate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized) . After incubation in this solution for 12 hours the protein was dialyzed to 10 mmolar sodium phosphate ( Figure 3) .
  • the 5' primer has the sequence 5' GCGCCCGGGATAAGCCAAGAACCAACG 3' and contains a Smal restriction enzyme site (underlined) followed by 21 nucleotides resembling an efficient signal for the initiation of translation in eukaryotic cells (J. Mol. Biol. 1987, 196. 947-950, Kozak, M.) .
  • the 3' primer has the sequence 5' GCGGGTACCT AAAATTTCAAGAAGGGCAC 3' and contains the cleavage site for the restriction endonuclease A ⁇ p718 and 21 nucleotide ⁇ complementary to the 3' non-tran ⁇ lated sequence of the hMutT2 gene.
  • the amplified sequences were isolated from a 1% agarose gel using a commercially available kit ("Geneclean, " BIO 101 Inc., La Jolla, Ca.) . The fragment was then digested with the endonuclease ⁇ Smal and A ⁇ p7l8 and then purified again on a 1% agarose gel. Thi ⁇ fragment i ⁇ designated F2.
  • the vector pA2 (modification of pVL941 vector, di ⁇ cussed below) is used for the expression of the hMutT2 protein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texa ⁇ Agricultural Experimental Station Bulletin No. 1555) .
  • Thi ⁇ expre ⁇ ion vector contain ⁇ the ⁇ trong polyhedrin promoter of the Autographa californica nuclear polyhedro ⁇ i ⁇ viru ⁇ (AcMNPV) followed by the recognition sites for the restriction endonuclease ⁇ Smal and A ⁇ p7l8.
  • the polyadenylation site of the simian virus (SV)40 is used for efficient polyadenylation.
  • the beta-galactosidase gene from E.coli i ⁇ inserted in the same orientation a ⁇ the polyhedrin promoter followed by the polyadenylation ⁇ ignal of the polyhedrin gene.
  • the polyhedrin ⁇ equences are flanked at both ⁇ ide ⁇ by viral ⁇ equence ⁇ for the cell-mediated homologou ⁇ recombination of cotransfected wild-type viral DNA.
  • Many other baculovirus vectors could be used in place of pRGl such as pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D. , Virology, 170:31-39).
  • the plasmid was digested with the restriction enzymes Smal and Asp718 and then depho ⁇ phorylated using calf intestinal phosphatase by procedures known in the art.
  • the DNA was then isolated from a 1% agarose gel using the commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.) .
  • Thi ⁇ vector DNA i ⁇ designated V2.
  • Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNA ligase.
  • E. coli HB101 cells were then transformed and bacteria identified that contained the plasmid (pBac hMutT2) with the hMutT2 gene using the enzymes Smal and Asp718. The sequence of the cloned fragment was confirmed by DNA sequencing.
  • plaque assay performed similar as described by Summers and Smith (supra) .
  • an agarose gel with "Blue Gal” (Life Technologies Inc., Gaithersburg) wa ⁇ used which allows an easy isolation of blue stained plaques.
  • a detailed description of a "plaque assay” can also be found in the user's guide for ineect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9- 10) .
  • Sf9 cell ⁇ were grown in Grace' ⁇ medium supplemented with 10% heat-inactivated FBS.
  • the cells were infected with the recombinant baculovirus V-hMutT2 at a multiplicity of infection (MOD of 2.
  • MOD multiplicity of infection
  • the medium wa ⁇ removed and replaced with SF900 II medium minu ⁇ methionine and cy ⁇ teine (Life Technologie ⁇ Inc. , Gaither ⁇ burg) .
  • 42 hour ⁇ later 5 ⁇ Ci of 35 S-methionine and 5 ⁇ Ci 35 S cysteine (A ersham) were added.
  • the cells were further incubated for 16 hours before they were harve ⁇ ted by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.
  • hMutT2 HA The expression of plasmid, hMutT2 HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E. coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site.
  • the HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously described (I.
  • the PCR product contains a EcoRI ⁇ ite, hMutT2 coding ⁇ equence followed by HA tag fu ⁇ ed in frame, a translation termination stop codon next to the HA tag, and an Xhol site.
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp were digested with EcoRI and Xhol restriction enzyme and ligated.
  • the ligation mixture was transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture was plated on ampicillin media plates and resistant colonies were selected. Plasmid DNA was isolated from transformant ⁇ and examined by restriction analysi ⁇ for the presence of the correct fragment.
  • hMutT2 For expression of the recombinant hMutT2, COS cells were transfected with the expression vector by DEAE- DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). The expression of the hMutT2 HA protein was detected by radiolabelling and immunoprecipitation method (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells were labelled for 8 hours with 35 S-cysteine two days post transfection.
  • Northern blot analysi ⁇ wa ⁇ carried out to examine the levels of expres ⁇ ion of hMutT2 in human ti ⁇ ues.
  • Total cellular RNA samples were isolated with RNAzolTM B sy ⁇ tem (Biotecx Laboratorie ⁇ , Inc. 6023 South Loop Ea ⁇ t, Hou ⁇ ton, TX 77033) .
  • the labeling reaction wa ⁇ done according to the Stratagene Prime- It kit with 50ng DNA fragment.
  • the labeled DNA wa ⁇ purified with a Select-G-50 column. (5 Prime - 3 Prime, Inc. 5603 Arapahoe Road, Boulder, CO 80303) .
  • the filter wa ⁇ then hybridized with radioactive labeled full length hMutT2 gene at 1,000,000 cpm/ml in 0.5 M NaP0 4 , pH 7.4 and 7% SDS overnight at 65 * C.
  • the filter wa ⁇ then wa ⁇ hed twice at room temperature and twice at 60 * C with 0.5 x SSC, 0.1% SDS, and then exposed at -70 * C overnight with an intensifying screen.
  • the me ⁇ age RNA for hMutT2 i ⁇ abundant in thymu ⁇ , te ⁇ tis, gall bladder, kidney, liver, lung, spleen, prostate, placenta ( Figure 4) .
  • ADDRESSEE CARELLA, BYRNE, BAIN, GILFILLAN,
  • AACAGTTCCG ACCACCAATG GGGGGCTACT GCATAGAGTT CCCTGCAGGT CTCATAGATG 420
  • Tyr lie lie lie Ser Glu Glu Leu lie Ser Glu Gly Ly ⁇ Trp Val Ly ⁇
  • Cys lie Glu Phe Pro Ala Gly Leu lie Asp Asp Gly Glu Thr Pro
  • Gly Asp lie Ala Glu Cys Ser Pro Ala Val Cys Met Asp Pro Gly

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Abstract

On décrit un polypeptide d'origine humaine appelé hMutT2 et l'ADN (ARN) codant ce polypeptide, ainsi qu'un procédé pour le préparer par des techniques de recombinaison. On décrit également des procédés utilisant ce polypeptide pour hydrolyser et éliminer les nucléotides contenant une guanine oxydée dans un pool de nucléotides, afin d'assurer une synthèse correcte de l'ADN. On décrit également des techniques diagnostiques qui permettent de détecter la présence d'une forme mutée de la protéine hMutT2 ou de détecter une surexpression de cette protéine.
PCT/US1994/013187 1994-11-15 1994-11-15 MutT2 HUMAIN WO1996015222A1 (fr)

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JP8516005A JPH10509320A (ja) 1994-11-15 1994-11-15 ヒトＭｕｔＴ２
EP95903538A EP0795006A4 (fr) 1994-11-15 1994-11-15 MutT2 HUMAIN
PCT/US1994/013187 WO1996015222A1 (fr) 1994-11-15 1994-11-15 MutT2 HUMAIN
AU12558/95A AU1255895A (en) 1994-11-15 1994-11-15 Human mutt2
US08/916,989 US6103871A (en) 1994-11-15 1997-08-21 Human mutT2
US09/432,253 US6344547B1 (en) 1994-11-15 1999-11-02 Human MutT2
US09/974,800 US6552174B2 (en) 1994-11-15 2001-10-12 Human MutT2 antibodies

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Cited By (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)

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 268, No. 31, issued 05 November 1993, K. SAKUMI et al., "Cloning and Expression of a cDNA for a Human Enzyme that Hydrolyzes 8-Oxo-dGTP, a Mutagenic Substrate for DNA Synthesis", pages 23524-23530. *
MOLECULAR AND GENERAL GENETICS, Volume 206, issued 1987, M. AKIYAMA et al., "Molecular Cloning and Nucleotide Sequence of the MutT Mutator of Escherichia Coli that Causes A:T to C:G Transversion", pages 9-16. *
MUTATION RESEARCH, Volume 299, issued 1993, J. TCHOU et al., "Repair of DNA Containing the Oxidatively-Damagea Base, 8-Oxoguanine", pages 277-287. *
NATURE, Volume 355, issued 16 January 1992, H. MAKI et al., "MutT Protein Specifically Hydrolyses a Potent Mutagenic Substrate for DNA Synthesis", pages 273-275. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 89, issued November 1992, J. MO et al., "Hydrolytic Elimination of a Mutagenic Nucleotide, 8-OxodGTP, by Human 18-Kilodalton Protein: Sanitization of Nucleotide Pool", pages 11021-11025. *
See also references of EP0795006A4 *

Cited By (4)

* 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)
WO2000065056A3 (fr) * 1999-04-26 2001-03-01 Beth Israel Hospital ACIDES NUCLEIQUES CODANT UN POLYPEPTIDE COMPRENANT UN DOMAINE $i(MUTT)
US8153400B1 (en) 1999-04-26 2012-04-10 Beth Israel Deaconess Medical Center, Inc. Nucleic acids encoding a mutT domain-containing polypeptide
US8614300B2 (en) 1999-04-26 2013-12-24 Beth Israel Deconess Medical Center, Inc. Nucleic acids encoding a mut-T domain-containing polypeptide

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EP0795006A4 (fr) 1999-10-27

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