EP0783567A1 - Gene de la galactokinase humaine - Google Patents

Gene de la galactokinase humaine

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Publication number
EP0783567A1
EP0783567A1 EP95921460A EP95921460A EP0783567A1 EP 0783567 A1 EP0783567 A1 EP 0783567A1 EP 95921460 A EP95921460 A EP 95921460A EP 95921460 A EP95921460 A EP 95921460A EP 0783567 A1 EP0783567 A1 EP 0783567A1
Authority
EP
European Patent Office
Prior art keywords
seq
galactokinase
sequence
dna
nucleic acid
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP95921460A
Other languages
German (de)
English (en)
Other versions
EP0783567A4 (fr
Inventor
Derk Jon Bergsma
Dwight Edward Stambolian
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Pennsylvania Penn
SmithKline Beecham Corp
Original Assignee
University of Pennsylvania Penn
SmithKline Beecham Corp
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 University of Pennsylvania Penn, SmithKline Beecham Corp filed Critical University of Pennsylvania Penn
Publication of EP0783567A1 publication Critical patent/EP0783567A1/fr
Publication of EP0783567A4 publication Critical patent/EP0783567A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • This invention relates to human galactokinase and the identification of galactokinase mutations, a missense and nonsense, as well as isolated nucleic acids encoding same, recombinant host cell transformed with DNA encoding such proteins and to uses of the expressed proteins and nucleic acid sequences in therapeutic and diagnostic applications.
  • Galactokinase deficiency is one of three known forms of galactosemia. The other forms are galactose-1 -phosphate uridyltransf erase deficiency and UDP- galactose-4-epimerase deficiency. All three enzymes are involved in galactose metabolism, i.e., the conversion of galactose to glucose in the body. Galactokinase deficiency is inherited as an autosomal recessive trait with a heterozygote frequency estimated to be 0.2% in the general population (see, e.g., Levy et al., J. Pediatr.. £2:871-877 (1978)).
  • Galactokinase activity has been found in a variety of mammalian tissues, including liver, kidney, brain, lens, placenta, erythrocytes and leukocytes. While the protein has been purified from E. coli, the purification of the protein from mammalian tissues has proven difficult due to its low cellular concentration. In addition, the molecular basis of galactokinase deficiency is unknown.
  • This invention provides a human galactokinase gene.
  • the DNAs of this invention such as the specific sequences disclosed herein, are useful in that they encode the genetic information required for expression of this protein. Additionally, the sequences may be used as probes in order to isolate and identify additional members, of the family, type and/or subtype as well mutations which may form the basis of galactokinase deficiency which may be characterized by site-specific mutations or by atypical expression of the galactokinase gene.
  • the galactokinase gene is also useful as a diagnostic agent to identify mutant galactokinase proteins or as a therapeutic agent via gene therapy.
  • This invention provides isolated nucleic acid molecules encoding human galactokinase, as well as nucleic acid molecules encoding missense and nonsense mutations, which includes mRNAs, DNAs (e.g., cDNA, genomic DNA, etc.), as well as antisense analogs thereof and diagnostically or therapeutically useful fragments thereof.
  • This invention also provides recombinant vectors, such as cloning and expression plasmids useful as reagents in the recombinant production of human galactokinase proteins, as well as recombinant prokaryotic and/or eukaryotic host cells comprising a human galactokinase nucleic acid sequence.
  • This invention also provides a process for preparing human galactokinase proteins which comprises culturing recombinant prokaryotic and/or eukaryotic host cells, containing a human galactokinase nucleic acid sequence, under conditions promoting expression of said protein and subsequent recovery thereof of said protein.
  • Another related aspect of this invention is isolated human galactokinase proteins produced by said method.
  • this invention also provides antibodies that are directed to (i.e., bind) human galactokinase proteins.
  • This invention also provides an isolated human galactokinase proteins having a missense or nonsense mutation and antibodies (monoclonal or polyclonal) that are specifically reactive with said proteins.
  • This invention also provides nucleic acid probes and PCR primers comprising nucleic acid molecules of sufficient length to specifically hybridize to human galactokinase sequences.
  • This invention also provides a method to diagnose human galactokinase deficiency which comprises isolating a nucleic acid sample from an individual and assaying the sequence of said nucleic acid sample with the reference gene of the invention and comparing differences between said sample and the nucleic acid of the instant invention, wherein said differences indicate mutations in the human galactokinase gene isolated from an individual.
  • the sample can be assayed by direct sequence comparison (i.e., DNA sequencing), wherein the sample nucleic acid can be compared to the reference galactokinase gene, by hybridization (e.g., mobility shift assays such as heteroduplex gel electrophoresis, SSCP or other techniques such as Northern or Southern blotting which are based upon the length of the nucleic acid sequence) or other known gel electrophoresis methods such as
  • the diagnostic method comprises isolating cells from an individual containing genomic DNA and assaying said sample (e.g., cellular RNA) by in situ hybridization using the DNA sequence of the invention, or at least one exon, or a fragment containing at least 15, preferably 18, and more preferably 21 contiguous base pairs as a probe.
  • This invention also provides an antisense oligonucleotide having a sequence capable of binding with mRNAs encoding human galactokinase so as to identify mutant galactokinase genes.
  • This invention also provides yet another method to diagnose human galactokinase deficiency which comprises obtaining a serum or tissue sample; allowing such sample to come in contact with an antibody or antibody fragment which specifically binds to a mutant human galactokinase protein of the invention under conditions such that an antigen-antibody complex is formed between said antibody (or antibody fragment) and said mutant galactokinase protein; and detecting the presence or absence of said complex.
  • This invention also provides transgenic non-human animals comprising a nucleic acid molecule encoding human galactokinase. Also provided are methods for use of said transgenic animals as models for disease states, mutation and SAR.
  • This invention also provides a method for treating conditions which are related to insufficient human galactokinase activity which comprises administering to a patient in need thereof a pharmaceutical composition containing the galactokinase protein of the invention which is effective to supplement a patient's endogenous galactokinase and thereby alleviating said condition.
  • This invention also provides a method for treating conditions which are related to insufficient human galactokinase activity via gene therapy.
  • An additional, or reference, gene comprising the non-mutant galactokinase gene of the instant invention is inserted into a patient's cells either in vivo or ex vivo.
  • the reference gene is expressed in transfected cells and as a result, the protein encoded by the reference gene corrects the defect (i.e., galactokinase deficiency) thus permitting the transfected cells to function normally and alleviating disease conditions (or symptoms).
  • Figure 1 depicts the intron/exon organization of the human galactokinase gene.
  • Figure 2 is the genomic DNA sequence (and single letter amino acid abbreviations) for human galactokinase [SEQ ID NO: 7].
  • the bolded DNA sequence corresponds to the exon regions whereas the normal or unbolded type corresponds to the intron regions of human galactokinase.
  • This invention relates to human galactokinase (amino acid and nucleotide sequences) and its use as a diagnostic and therapeutic.
  • the particular cDNA and amino acid sequence of human galactokinase is identified by SEQ ID NO:4 as described more fully below.
  • This invention also relates to the genomic DNA sequence for human galactokinase [SEQ ID NO: 7] and also to mutant human galactokinase genes and amino acid sequences [SEQ ID NO:5 and 6] and their use for diagnostic purposes.
  • SEQ ID NO: 7 genomic DNA sequence for human galactokinase
  • mutant human galactokinase genes and amino acid sequences [SEQ ID NO:5 and 6] and their use for diagnostic purposes.
  • the following additional terms will be employed, and are intended to be defined as indicated below.
  • an “antigen” refers to a molecule containing one or more epi topes that will stimulate a host's immune system to make a humoral and or cellular antigen-specific response.
  • the term is also used herein interchangeably with "immunogen.”
  • epitope refers to the site on an antigen or hapten to which a specific antibody molecule binds.
  • the term is also used herein interchangeably with “antigenic determinant” or “antigenic determinant site.”
  • a coding sequence is "operably linked to" another coding sequence when RNA polymerase will transcribe the two coding sequences into a single mRNA, which is then translated into a single polypeptide having amino acids derived from both coding sequences.
  • the coding sequences need not be contiguous to one another so long as the expressed sequence is ultimately processed to produce the desired protein.
  • "Recombinant” polypeptides refer to polypeptides produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide.
  • Synthetic polypeptides are those prepared by chemical synthesis.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo: i.e., capable of replication under its own control.
  • a “vector” is a replicon, such as a plasmid, phage, or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a “replication-deficient virus” is a virus in which the excision and/or replication functions have been altered such that after transfection into a host cell, the virus is not able to reproduce and/or infect addition cells.
  • a “reference” gene refers to the galactokinase sequence of the invention and is understood to include the various sequence polymorphisms that exist, wherein nucleotide substitutions in the gene sequence exist, but do not affect the essential function of the gene product.
  • a “mutant" gene refers to galactokinase sequences different from the reference gene wherein nucleotide substitutions and/or deletions and/or insertions result in impairment of the essential function of the gene product such that the levels of galactose in an individual (or patient) are atypically elevated.
  • the G to A substitution at position 122 of human galactokinase [SEQ LD NO: 5] is a missense mutation associated with patients who are galactokinase deficient.
  • Another T for G substitution produces an in-frame nonsense codon at amino acid position 80 of the mature protein. The result is a truncated protein consisting of the first 79 amino acids of human galactokinase.
  • a DNA "coding sequence of or a "nucleotide sequence encoding" a particular protein is a DNA sequence which is transcribed and translated into a polypeptide when placed under the control of appropriate regulatory sequences.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bound at the 3' terminus by a translation start codon (e.g., ATG) of a coding sequence and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes.
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.
  • control sequences refers collectively to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the expression (i.e., the transcription and translation) of a coding sequence in a host cell.
  • a control sequence "directs the expression" of a coding sequence in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.
  • a “host cell” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous DNA sequence.
  • a cell has been "transformed” by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane.
  • Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the exogenous DNA may be maintained on an episomal element, such as a plasmid.
  • a stably transformed or transfected cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cell containing the exogenous DNA.
  • Transfection refers to a process by which cells take up foreign DNA and integrate that foreign DNA into their chromosome. Transfection can be accomplished, for example, by various techniques in which cells take up DNA (e.g., calcium phosphate precipitation, electroporation, assimilation of liposomes, etc.), or by infection, in which viruses are used to transfer DNA into cells.
  • various techniques in which cells take up DNA e.g., calcium phosphate precipitation, electroporation, assimilation of liposomes, etc.
  • viruses are used to transfer DNA into cells.
  • a "target cell” is a cell(s) that is selectively transfected over other cell types (or cell lines).
  • a “clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • a "heterologous" region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature. Thus, when the heterologous region encodes a gene, the gene will usually be flanked by DNA that does not flank the gene in the genome of the source animal.
  • Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene). Allelic variation or naturally occurring mutational events do not give rise to a heterologous region of DNA, as used herein.
  • Constants which are related to insufficient human galactokinase activity or a "deficiency in galactokinase activity” means mutations of the galactokinase protein which affects galactokinase activity or may affect expression of galactokinase or both such that the levels of galactose in a patient are atypically elevated.
  • this definition is intended to cover atypically low levels of galactokinase expression in a patient due to defective control sequences for the reference galactokinase protein.
  • This invention provides an isolated nucleic acid molecule encoding a human galactokinase protein and substantially similar sequences.
  • Isolated nucleic acid sequences are "substantially similar” if: (i) they are approximately the same length (i.e., at least 80% of the coding region of SEQ ID NO:4); (ii) they encode a protein with the same (i.e., within an order of magnitude) galactokinase activity as the protein encoded by SEQ ID NO:4; and (iii) they are capable of hybridizing under moderately stringent conditions to SEQ ID NO:4; or they encode DNA sequences which are degenerate to SEQ ID NO:4. Degenerate DNA sequences encode the same amino acid sequence as SEQ ID NO:4, but have variation(s) in the nucleotide coding sequences. Hybridization under moderately stringent conditions is outlined below.
  • Hybridization under moderately stringent conditions can be performed as follows. Nitrocellulose filters are prehybridized at 65°C in a solution containing 6X SSPE, 5X Denhardt's solution (lOg Ficoll, lOg BSA and lOg Polyvinylpyrrolidone per liter solution), 0.05% SDS and 100 micrograms tRNA. Hybridization probes are labeled, preferably radiolabelled (e.g., using the Bios TAG-IT® kit). Hybridization is then carried out for approximately 18 hours at 65°C. The filters are then washed in a solution of 2X SSC and 0.5% SDS at room temperature for 15 minutes (repeated once). Subsequently, the filters are washed at 58°C, air-dried and exposed to X-ray film overnight at -70°C with an intensifying screen.
  • substantially similar sequences are substantially the same when about 66% (preferably about 75%, and most preferably about 90%) of the nucleotides or amino acids match over a defined length (i.e., at least 80% of the coding region of SEQ ID NO:4) of the molecule and the protein encoded by such sequence has the same (i.e., within an order of magnitude) galactokinase activity as the protein encoded by SEQ ID NO:4.
  • substantially similar refers to the sequences having similar identity to the sequences of the instant invention.
  • nucleotide sequences that are substantially the same can be identified by hybridization or by sequence comparison.
  • Protein sequences that are substantially the same can be identified by one or more of the following: proteolytic digestion, gel electrophoresis and/or microsequencing.
  • This invention also provides isolated nucleic acid molecules encoding a missense mutation (SEQ ID NO:5) or a nonsense mutation (SEQ ID NO:6) of the human galactokinase protein and DNA sequences which are degenerate to SEQ ID NO:5 or 6.
  • Degenerate DNA sequences encode the same amino acid (or termination site) sequence as SEQ ID NO:5 or 6, but have variation(s) in the nucleotide coding sequences.
  • One means for isolating a nucleic acid molecule encoding for a human galactokinase is to probe a human genomic or cDNA library with a natural or artificially designed probe using art recognized procedures (See for example: "Current Protocols in Molecular Biology", Ausubel, F.M., et al. (eds.) Greene Publishing Assoc. and John Wiley Interscience, New York, 1989,1992). It is appreciated to one skilled in the art that SEQ ID NO:4, or fragments thereof (comprising at least 15 contiguous nucleotides), is a particularly useful probe.
  • probes for this purpose are set forth in Table 1, or hybridizable fragments thereof (i.e., comprising at least 15 contiguous nucleotides). It is also appreciated that such probes can be and are preferably labeled with an analytically detectable reagent to facilitate identification of the probe.
  • Useful reagents include but are not limited to radioactivity, fluorescent dyes or enzymes capable of catalyzing the formation of a detectable product.
  • the probes are thus useful to isolate complementary copies of genomic DNA, cDNA or RNA from human, mammalian or other animal sources or to screen such sources for related sequences (e.g., additional members of the family, type and/or subtype) and including transcriptional regulatory and control elements defined above as well as other stability, processing, translation and tissue specificity-determining regions from 5' and/or 3' regions relative to the coding sequences disclosed herein.
  • related sequences e.g., additional members of the family, type and/or subtype
  • transcriptional regulatory and control elements defined above as well as other stability, processing, translation and tissue specificity-determining regions from 5' and/or 3' regions relative to the coding sequences disclosed herein.
  • Gene therapy means gene supplementation. That is, an additional (i.e., reference) copy of the gene of interest is inserted into a patients' cells. As a result, the protein encoded by the reference gene corrects the defect (i.e., galactokinase deficiency) and permits the cells to function normally thus alleviating disease symptoms.
  • Gene therapy of the present invention can occur in vivo or ex vivo.
  • Ex vivo gene therapy requires the isolation and purification of patient cells, the introduction of a therapeutic gene, and introduction of the genetically altered cells back into the patient.
  • a replication-deficient virus such as a modified retrovirus can be used to introduce the therapeutic gene (galactokinase) into such cells.
  • MMLV mouse Moloney leukemia virus
  • MMLV mouse Moloney leukemia virus
  • the therapeutic gene is typically "packaged” for administration to a patient such as in liposomes or in a replication-deficient virus such as adenovirus (see, e.g., Berkner, K.L., Curr. Top. Microbiol. Immunol..15_8_:39-66 (1992)) or adeno-associated virus (AAV) vectors (see, e.g., Muzyczka, N., Curr. Top. Microbiol. Immunol.. JL5£:97-129 (1992) and U.S. Patent 5,252,479 "Safe Vector for Gene Therapy”).
  • adenovirus see, e.g., Berkner, K.L., Curr. Top. Microbiol. Immunol..15_8_:39-66 (1992)
  • AAV adeno-associated virus
  • Another approach is administration of so-called “naked DNA” in which the therapeutic gene is directly injected into the bloodstream or muscle tissue.
  • Cell types useful for gene therapy of the present invention include hepatocytes, f ⁇ broblasts, lymphocytes, any cell of the eye (e.g., retina), epithelial and endothelial cells.
  • the cells are hepatocytes, any cell of the eye or respiratory (or pulmonary) epithelial cells. Transfection of (pulmonary) epithelial /09374 PCMJS95/06743
  • cells can occur via inhalation of a neubulized preparation of DNA vectors in liposomes, DNA-protein complexes or replication-deficient adenoviruses (see, e.g., U.S. Patent 5,240,846 "Gene Therapy Vector for Cystic Fibrosis".
  • This invention also provides for a process to prepare human galactokinase proteins.
  • Non-mutant proteins are defined with reference to the amino acid sequence listed in SEQ ID NO:4 and includes variants with a substantially similar amino acid sequence that have the same galactokinase activity.
  • Additional proteins of this invention include mutant human galactokinase proteins as set forth in SEQ ID NO: 5 or 6.
  • the proteins of this invention are preferably made by recombinant genetic engineering techniques.
  • the isolated nucleic acids particularly the DNAs can be introduced into expression vectors by operatively linking the DNA to the necessary expression control regions (e.g., regulatory regions) required for gene expression.
  • the vectors can be introduced into the appropriate host cells such as prokaryotic (e.g., bacterial), or eukaryotic (e.g., yeast or mammalian) cells by methods well known in the art (Ausubel et al., supra).
  • the coding sequences for the desired proteins having been prepared or isolated can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells which they can transform include, but is not limited to, the bacteriophage ⁇ (E. coli . pBR322 (£. coli).
  • pACYC177 (£. coli).
  • pKT230 (gram-negative bacteria), pGVl 106 (gram-negative bacteria), pLAFRl (gram-negative bacteria), pME290 (non-E. £ ⁇ _H gram-negative bacteria), pHV14 (£. £pJi and Bacillus subtilis).
  • pBD9 Bacillus subtilis.
  • pIJ61 Streptomyces
  • pUC6 Streptomyces
  • YIp5 Sacharornyces
  • YCpl9 Sacharornyces
  • the gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction.
  • the coding sequence may or may not contain a signal peptide or leader sequence.
  • the subunit antigens of the present invention can be expressed using, for example, the £. coli tac promoter or the protein A gene (spa) promoter and signal sequence. Leader sequences can be removed by the bacterial host in post-translational processing. S_££, £ * &., U.S. Patent Nos.
  • regulatory sequences which allow for regulation of the expression of the protein sequences relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
  • An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the "control" of the control sequences (i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence).
  • control i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • Modification of the sequences encoding the particular antigen of interest may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame.
  • control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above.
  • a vector such as the cloning vectors described above.
  • the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
  • Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art. ⁇ e., ej ⁇ , T. Maniatis et al., supra; DNA Cloning. Vols. I and II, supra; Nucleic Acid Hybridization, supra.
  • a number of prokaryotic expression vectors are known in the art. ⁇ £ £ , e ⁇ , U.S. Patent Nos. 4,578,355; 4,440,859; 4,436,815; 4,431,740; 4,431,739; 4,428,941; 4,425,437; 4,418,149; 4,411,994; 4,366,246; 4,342,832; ss& 2 Q U.K. Patent Applications GB 2,121,054; GB 2,008,123; GB 2,007,675; and European Patent Application 103,395. Yeast expression vectors are also known in the art. See, e.g.. U.S. Patent Nos.
  • pSV2neo (as described in J. Mol. Appl. Genet. 1 :327-341) which uses the S V40 late promoter to drive expression in mammalian cells or pCDNAlneo, a vector derived from pCDNAl (Mol. Cell Biol. 7:4125-29) which uses the CMV promoter to drive expression. Both these latter two vectors can be employed for transient or stable (using G418 resistance) expression in mammalian cells. Insect cell expression systems, e.g., Drosophila.
  • the proteins of the present invention are produced by growing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed.
  • Preferred mammalian cells include human embryonic kidney cells, monkey kidney (HEK-293cells), fibroblast (COS) cells, Chinese hamster ovary (CHO) cells, Drosophila or murine L-cells. If the expression system secretes the protein into growth media, the protein can be purified directly from the media. If the protein is not secreted, it is isolated from cell lysates or recovered from the cell membrane fraction.
  • An alternative method to identify proteins of the present invention is by constructing gene libraries, using the resulting clones to transform J coli and pooling and screening individual colonies using polyclonal serum or monoclonal antibodies to galactokinase.
  • the proteins of the present invention may also be produced by chemical synthesis such as solid phase peptide synthesis, using known amino acid sequences or amino acid sequences derived from the DNA sequence of the genes of interest. Such methods are known to those skilled in the art. Chemical synthesis of peptides is not particularly preferred.
  • the proteins of the present invention or their fragments comprising at least one epitope can be used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal, (e.g., mouse, rabbit, goat, horse, etc.) is immunized with the protein of the present invention, or a fragment thereof, capable of eliciting an immune response (i.e., having at least one epitope). Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies is used, the polyclonal antibodies can be purified by immunoaffinity chromatography or other known procedures.
  • Monoclonal antibodies to the proteins of the present invention, and to the fragments thereof, can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by using hybridoma technology is well known.
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. ⁇ S ⁇ . e.g.. M.
  • monoclonal antibodies specifically reactive with mutant galactokinase proteins, e.g., the missense mutation of SEQ ID NO:5 or nonsense mutation of SEQ ID NO:6.
  • Monoclonal antibodies are useful in purification, using immunoaffinity techniques, of the individual antigens which they are directed against.
  • genes encoding the monoclonals of interest may be isolated from the hybridomas by PCR techniques known in the art and cloned and expressed in the appropriate vectors.
  • the antibodies of this invention, whether polyclonal or monoclonal have additional utility in that they may be employed reagents in immunoassays, RIA, ELISA, and the like.
  • “monoclonal antibody” is understood to include antibodies derived from one species (e.g., murine, rabbit, goat, rat, human, etc.) as well as antibodies derived from two (or perhaps more) species (e.g., chimeric and humanized antibodies). Chimeric antibodies, in which non-human variable regions are joined or fused to human constant regions (see, e.g. Liu et al., Proc. Natl Acad. Sci. USA. 84:3439 (1987)), may also be used in assays or therapeutically. Preferably, a therapeutic monoclonal antibody would be "humanized” as described in Jones et al., Nature.
  • this invention also contemplates antibodies, polyclonal or monoclonal (including chimeric and "humanized") directed to epitopes corresponding to amino acid sequences disclosed herein from human galactokinase. Methods for the production of polyclonal and monoclonal antibodies are well known, see for example Chap. 11 of Ausubel et al. (supra).
  • the antibody When the antibody is labeled with an analytically detectable reagent such a radioactivity, fluorescence, or an enzyme, the antibody can be use to detect the presence or absence of human galactokinase and/or its quantitative level.
  • antibodies (polyclonal or monoclonal) specific for the missense and nonsense mutations of the present invention are useful for diagnostic purposes.
  • a serum or tissue sample e.g., liver, lung, etc.
  • an antibody or antibody fragment which specifically binds to a mutant human galactokinase protein of the invention under conditions such that an antigen- antibody complex is formed between said antibody (or antibody fragment) and said mutant galactokinase protein.
  • the detection for the presence or absence of said complex is within the skill of the art (e.g., ELISA, RIA, Western Blotting, Optical Biosensor (e.g., BIAcore - Pharmacia Biosensor, Uppsala, Sweden) and do not limit this invention.
  • compositions comprising an effective amount of the galactokinase protein of the invention and a pharmaceutically acceptable carrier.
  • Pharmaceutical compositions of proteinaceous drugs of this invention are particularly useful for parenteral administration, i.e., subcutaneously, intramuscularly or intravenously.
  • the galactokinase protein is surrounded by a membrane bound vesicle, such as a liposome.
  • compositions for parenteral administration will commonly comprise a solution of the compounds of the invention or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier.
  • an acceptable carrier preferably an aqueous carrier.
  • aqueous carriers may be employed, e.g., water, buffered water, 0.4% saline, 0.3% glycine, and the like. These solutions are sterile and generally free of particulate matter. These solutions may be sterilized by conventional, well known sterilization techniques.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc.
  • concentration of the compound of the invention in such pharmaceutical formulation can very widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected.
  • a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain 1 mL sterile buffered water, and 50 mg of a compound of the invention.
  • a pharmaceutical composition of the invention for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 150 mg of a compound of the invention.
  • Actual methods for preparing parenterally administrable compositions are well known or will be apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science. 15th ed., Mack Publishing Company, Easton, Pennsylvania.
  • the compounds described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional proteins and art-known lyophilization and reconstitution techniques can be employed.
  • the physician will determine the dosage of the present therapeutic agents which will be most suitable and it will vary with the form of administration and the particular compound chosen, and furthermore, it will vary with the particular patient under patient under treatment. He will generally wish to initiate treatment with small dosages substantially less than the optimum dose of the compound and increase the dosage by small increments until the optimum effect under the circumstances is reached. It will generally be found that when the composition is administered orally, larger quantities of the active agent will be required to produce the same effect as a smaller quantity given parenterally.
  • the therapeutic dosage will generally be from 1 to 10 milligrams per day and higher although it may be administered in several different dosage units.
  • the pharmaceutical composition of the invention can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the disease and its complications.
  • compositions containing the present compounds or a cocktail thereof are administered to a patient not already in a disease state to enhance the patient's resistance.
  • Single or multiple administrations of the pharmaceutical compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • the pharmaceutical composition of the invention should provide a quantity of the compounds of the invention sufficient to effectively treat the patient.
  • This invention also contemplates use of the galactokinase genes of the instant invention as a diagnostic. For example, some diseases result from inherited defective genes. These genes can be detected by comparing the sequence of the defective gene with that of a normal one. Subsequently, one can verify that a "mutant" gene is associated with galactokinase deficiency by measurement of galactose. That is, a mutant gene would be associated with (atypically) elevated levels of galactose in a patient.
  • RNA from an individual can be transcribed with reverse transcriptase to cDNA which can then be amplified by polymerase chain reaction (PCR), cloned into an E. coli expression vector, and transformed into a galactokinase-deficient strain of E. coli.
  • PCR polymerase chain reaction
  • galactokinase-deficient cells will produce colonies that are white in color, whereas cells that have been transformed/complemented with a functional galactokinase gene will be red (see, e.g., Examples section). If most to all of the colonies from an individual are red, then the individual is considered to be normal with respect to galactokinase activity. If approximately 50% of the colonies are red (the other 50% white), then that individual is likely to be a carrier for galactokinase deficiency. If most to all of the colonies are white, then that individual is likely to be galactokinase deficient. Once "mutant" genes have been identified, one can then screen the population for carriers of the "mutant" galactokinase gene.
  • a carrier is a person in apparent health whose chromosomes contain a "mutant" galactokinase gene that may be transmitted to that person's offspring.
  • monoclonal antibodies that are specific for the mutant galactokinase proteins can be used for diagnostic purposes as described above. Individuals carrying mutations in the human galactokinase gene may be detected at the DNA level by a variety of techniques. Nucleic acids used for diagnosis (genomic DNA, mRNA, etc.) may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy (e.g., chorionic villi sampling or removal of amniotic fluid cells), and autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR, ligase chain reaction (LCR), strand displacement amplification (SDA), etc. (see, e.g., Saiki et al., Nature.224:163-166 (1986), Bej, et al., Crit. Rev. Biochem. Molec. Biol..26:301- 334 (1991), Birkenmeyer et al., J. Virol. Meth..25:117-126 (1991), Van Brunt, J., Bio Technologv. &:291-294 (1990)) prior to analysis.
  • RNA may also be used for the same purpose.
  • RNA can be reverse-transcribed and amplified at one time with PCR-RT (polymerase chain reaction - reverse transcriptase) or reverse- transcribed to an unamplified cDNA.
  • PCR primers complementary to the nucleic acid of the instant invention can be used to identify and analyze galactokinase mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal galactokinase genotype.
  • Point mutations can be identified by hybridizing amplified DNA to radiolabeled galactokinase RNA (of the invention) or alternatively, radiolabelled galactokinase antisense DNA sequences (of the invention).
  • Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures (Tm). Such a diagnostic would be particularly useful for prenatal and even neonatal testing.
  • point mutations and other sequence differences between the reference gene and "mutant" genes can be identified by yet other well-known techniques, e.g., direct DNA sequencing, single-strand conformational polymorphism (SSCP; Orita et al., Genomics.5:874-879 (1989)).
  • SSCP single-strand conformational polymorphism
  • a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures with radiolabeled nucleotides or by automatic sequencing procedures with fluorescent-tags.
  • Cloned DNA segments may also be used as probes to detect specific DNA segments.
  • the sensitivity of this method is greatly enhanced when combined with PCR.
  • the presence of nucleotide repeats may correlate to a change in galactokinase activity (causative change) or serve as marker for various polymorphisms.
  • DNA sequence differences may be achieved 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 by high resolution gel electrophoresis. DNA fragments of different sequences may 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 al., Science. 23_Q_:1242 (1985)).
  • sequence alterations in particular small deletions, may be detected as changes in the migration pattern of DNA heteroduplexes in non-denaturing gel electrophoresis (i.e., heteroduplex electrophoresis) (see, e.g., Nagamine et al., Am. J. Hum. Genet.. 45:337-339 (1989)).
  • Sequence changes at specific locations may 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. Natl. Acad. Sci. USA. 85:4397-4401 (1985)).
  • nuclease protection assays such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci. USA. 85:4397-4401 (1985)).
  • the detection of a specific DNA sequence may be achieved by methods such as hybridization (e.g., heteroduplex electroporation, see, White et al., Genomics.12:301-306 (1992), RNAse protection (e.g., Myers et al., Science. 22 ⁇ : 1242 (1985)) chemical cleavage (e.g., Cotton et al., Proc. Natl. Acad. Sci. USA.
  • hybridization e.g., heteroduplex electroporation, see, White et al., Genomics.12:301-306 (1992
  • RNAse protection e.g., Myers et al., Science. 22 ⁇ : 1242 (1985)
  • chemical cleavage e.g., Cotton et al., Proc. Natl. Acad. Sci. USA.
  • restriction enzymes e.g., restriction fragment length polymorphisms (RFLP) in which variations in the number and size of restriction fragments can indicate insertions, deletions, presence of nucleotide repeats and any other mutation which creates or destroys an endonuclease restriction sequence.
  • Southern blotting of genomic DNA may also be used to identify large (i.e., greater than 100 base pair) deletions and insertions.
  • mutations e.g., microdeletions, aneuploidies, translocations, inversions
  • in situ analysis See, e.g., Keller et al., DNA Probes, 2nd Ed., Stockton Press, New York, N.Y., USA (1993)). That is, DNA (or RNA) sequences in cells can be analyzed for mutations without isolation and/or immobilization onto a membrane.
  • Fluorescence in situ hybridization FISH is presently the most commonly applied method and numerous reviews of FISH have appeared.
  • some diseases are a result of, or are characterized by, changes in gene expression which can be detected by changes in the mRNA.
  • the galactokinase gene can be used as a reference to identify individuals expressing a decreased level of galactokinase, e.g., by Northern blotting or in situ hybridization. Defining appropriate hybridization conditions is within the skill of the art.
  • probes can vary widely but it is preferred that the probe be at least 15 nucleotides in length. It is also appreciated that such probes can be and are preferably labeled with an analytically detectable reagent to facilitate identification of the probe.
  • Useful reagents include but are not limited to radioactivity, fluorescent dyes or enzymes capable of catalyzing the formation of a detectable product. As a general rule the more stringent the hybridization conditions the more closely related genes will be that are recovered.
  • antisense oligonucleotides predicated upon the sequences disclosed herein for human galactokinase.
  • Synthetic oligonucleotides or related antisense chemical structural analogs are designed to recognize and specifically bind to a target nucleic acid encoding galactokinase and galactokinase mutations.
  • the general field of antisense technology is illustrated by the following disclosures which are incorporated herein by reference for purposes of background (Cohen, J.S., Trends in Pharm. Sci.. 10:435(1989) and Weintraub, H.M. Scientific American. Jan.(1990) at page 40).
  • Transgenic, non-human, animals may be obtained by transfecting appropriate fertilized eggs or embryos of a host with nucleic acids encoding human galactokinase disclosed herein, see for example U.S. Patents 4,736,866; 5,175,385; 5,175,384 and 5,175,386.
  • the resultant transgenic animal may be used as a model for the study of galactokinase.
  • useful transgenic animals are those which display a detectable phenotype associated with the expression of the receptor. Drugs may then be screened for their ability to reverse or exacerbate the relevant phenotype.
  • This invention also contemplates operatively linking the receptor coding gene to regulatory elements which are differentially responsive to various temperature or metabolic conditions, thereby effectively turning on or off the phenotypic expression in response to those conditions.
  • Galactokinase was obtained from human placenta as described by Stambolian et al. (Biochim Biophys Acta. £21:306-312 (1985)), which is incoiporated by reference in its entirety. In essence, human placenta tissue (obtained within 1 hour of parturition) was homogenized, centrifuged and the resulting supernatant was absorbed onto DEAE-Sephacel®. The material was eluted, precipitated with ammonium sulfate and then run through a sizing column (Sephadex G-100 SF®). Pooled active fractions were concentrated.
  • Purified protein was obtained following separation by SDS polyacrylamide electrophoresis and then Western blotted using standard techniques (see, Laemmli, Nature.222:680-685 (1970), or LeGendre et al., Biotechniques. 6:154 (1988)). Minute amounts of galactokinase were isolated (micrograms) from multiple rounds of protein purification. After a trypsin peptide digest, 7 peptide sequences were eventually isolated and identified. The three longest fragments are presented below: [SEQ ID NO: 1] Val Asn Leu He Gly Glu His Thr Asp Tyr Asn Gin Gly Leu Val Leu- Pro Met Ala Leu Glu Leu Met Thr Val Leu Val Gly Ser Pro Arg
  • Gly lie Met Asp Gin Phe He Ser Leu Met Gly Gin Lys /09374 PCMJS95 06743
  • the fragments were compared with peptide sequences encoded by cDNAs, in which the cDNAs were partially sequenced.
  • the cDNAs also known as expressed sequence tags or ESTs
  • the best alignments occurred with an EST sequence from a human osteoclastoma stromal cell library (SEQ ID NO:l showed 100% identity over 18 contiguous amino acids) and an EST sequence from a human pituitary library (SEQ ID NO:2 showed 95.5% identity over 22 contiguous amino acids).
  • SEQ ID NO:4 A full-length cDNA from the human osteoclastoma stromal cell library was identified and sequenced (SEQ ID NO:4) in its entirety on an automated ABI 373A Sequencer. Sequencing was confirmed on both strands. The corresponding amino acid sequence (SEQ ID NO:4) was compared against the peptide fragments identified above.
  • SEQ ID NO:l corresponds to amino acids 38-68 of the full-length human galactokinase protein.
  • SEQ ID NOs: 2 and 3 correspond to amino acids 367-388 and 167-195, respectively, of human galactokinase.
  • SEQ ID NO:4 was hybridized against a Northern blot containing human messenger RNA from placenta, brain, skeletal muscle, kidney, intestine, heart, lung and liver according to standard procedures (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory Press, 1989). Hybridization was strongest with human liver and lung tissue.
  • SEQ ID NO:4 was subcloned into an E. coli vector, plasmid pBluescript [Stratagene].
  • E. coli vector plasmid pBluescript [Stratagene].
  • C600K- a galactokinase-deficient strain
  • the transformed E. coli grew on MacConkey agar plates containing 1% galactose (and ampicillin @ 50ug/ml for plasmid selection), and produced brick red colonies, indicating sugar fermentation.
  • the red color is due to the action of acids, produced by galactose fermentation, upon bile salts and the indicator (neutral red) in MacConkey medium.
  • SEQ ID NO:4 was also subcloned into COS-1 cells [ATCC CRL 1650]. The cells were transfected, grown, and cell lysates were prepared. The lysates were assayed by a ,4 C galactokinase assay as described by Stambolian et al. (Exp. Eve Res.. 2£:231-237 (1984)) which is hereby incorporated by reference in its entirety. When expressed in transiently transfected COS cells, galactokinase activity was tenfold higher than control levels (6600 vs. 640 counts per minute - repeated three times). These results definitively confirm that SEQ ID NO:4 encodes a full-length, biologically active, human galactokinase gene.
  • the nucleic acid molecule of the invention can also be subcloned into an expression vector to produce high levels of human galactokinase (either fused to another protein, e.g., operatively linked at the 5' end with another coding sequence, or unfused) in transfected cells.
  • the expression vector would optionally encode a neomycin resistance gene to select for transfectants on the basis of ability to grow in G418 and a dihydrofolate reductase gene which permits amplification of the transfected gene in DHFR" cells.
  • the plasmid can then be introduced into host cell lines e.g., CHO ACC98, a nonadherent, DHFR" cell line adapted to grow in serum free medium, and human embryonic kidney 293 cells (ATCC CRL 1573), and transfected cell lines can be selected by G418 resistance.
  • host cell lines e.g., CHO ACC98, a nonadherent, DHFR" cell line adapted to grow in serum free medium, and human embryonic kidney 293 cells (ATCC CRL 1573), and transfected cell lines can be selected by G418 resistance.
  • a full-length galactokinase genomic gene coding region was identified from a lambda phage ( ⁇ Fix II) human genomic library (made from human placenta tissue) using the galK cDNA as a probe.
  • One isolate, designated clone 17 was deposited on
  • the genomic gene coding region is divided into at least 8 exons isolated from
  • GM00334 A fibroblast cell line (GM00334), derived from a patient with galactokinase deficiency, was obtained from the Coriell Institute for Medical research, 401 Haddon Ave., Camden, New Jersey, 08103. Total RNA was isolated from the cultured cells using the RNAZOL kit for isolation cf RNA (Biotecx, Houston, Tx). Cytoplasmic DNA (1 ug) was reversed transcribed with oligonucleotide primers 1823 [SEQ ID NO: 29] and 1825 [SEQ LD NO: 30]. The sample was amplified by 35 cycles at 94°C for 1 min., 60°C for 1 min. and 72°C for 7 min.
  • the DNA product was purified electrophoretically, ligated to the TA cloning vector (Invitrogen) and sequenced. Twelve cDNAs in total were sequenced (representing cloned PCR products of multiple independent PCR reactions). This procedure was also repeated with cultured fibroblasts from normal controls (i.e., persons not exhibiting galactokinase deficiency). A comparison with normal controls identified a single base substitution of A for G at position 122 of the "normal" human galactokinase gene [SEQ ID NO: 4). The result is a missense mutation in amino acid 32 from Val to Met [SEQ ID NO: 5].
  • the G to A base change creates a MscI endonuclease restriction site (i.e., TGG>tCCA) on the mutant allele.
  • This restriction site was then used to rapidly screen for the mutant allele in the parents of the patient with galactokinase deficiency.
  • the exon encoding galactokinase residues 1 to 5 i.e., exon 1, see Table 1 was cloned from a genomic lambda phage library and its DNA sequence was determined, including a portion of the flanking intron sequences.
  • Oligonucleotide primers (X2-50UT [SEQ ID NO: 31] and X2-30UT [SEQ ID NO: 32]) were designed to hybridize to intron sequences for the amplification of a 346 bp DNA fragment of the genomic DNA.
  • the PCR product was analyzed for the point mutation via RFLP, that is, the presence of a newly created MscI site as detected by electrophoresis of a 1.5% agarose gel. A "normal" allele remains uncut with the enzyme MscI, and thus migrates as a 346bp fragment on an agarose gel.
  • the PCR product from the patient with galactokinase deficiency (i.e., the G to A base change) is cleaved with MscI, resulting in two fragments of 193 and 153 bp, respectively.
  • the absence of 346 bp fragment indicates that the patient was homozygous for this allele.
  • PCR products from the parents of this patient, followed by a MscI digestion resulted in three fragments (346, 193 and 153 bp) which is consistent with a heterozygous pattern for the G to A base change. That is, the parents were both carriers of the same mutation.
  • a cDNA clone containing the G to A base change was subcloned into COS cells and assayed for galactokinase activity as previously described.
  • COS cells transfected with cDNA encoding the missense mutation had the same level of galactokinase activity as the host COS cells, namely 0.02 units/ug protein.
  • COS cells transfected with the non-mutant galactokinase cDNA [SEQ ID NO:4] had a fifty-fold higher activity compared to the host COS cells (i.e., control). This results supports the Val 32 to Met 32 substitution as the cause of the decreased enzymatic activity.
  • ADDRESSEE SmithKline Beecham Corp./Corporate Intellectual Property
  • B STREET: 709 Swedeland Road/UW2220
  • Val Asn Leu lie Gly Glu His Thr Asp Tyr Asn Gin Gly Leu Val Leu 1 5 10 15
  • MOLECULE TYPE protein
  • SEQUENCE DESCRIPTION SEQ ID NO : 2 :
  • GCT GCC AGG GAC CTG GTG AGC AAA GAG GGC TTC CGG CGG GCC CGG CAC 868
  • GCT GCC AGG GAC CTG GTG AGC AAA GAG GGC TTC CGG CGG GCC CGG CAC 868
  • MOLECULE TYPE DNA (genomic)
  • GCCTCGTCAT CTCCCCCATT GTAACTCCAC CCCAGGTCCT TGGAGACCAG CCTGGTGCCA 2340
  • CTACAGGCGC CCGCCACTAC GCCTGGCACA TTTTTTATAG TTCTAGTAGA GACTGGGGTT 3360
  • CACGCCCAGC CAATTTTTAT ATTTTTAGTA GAGACGGGGT TTCATGTTGG CCAGGCTGGC 3780
  • CTTCTTCTCC TCCTGCTCTG TGGTGGCCTC TTGAGGGCAG CACTCACCTT GGAAAGCATG 6360
  • GAGTGTTTCA ACCCTCACTG CTCCCTGAAG GACCAAGGTG TCCCATTTTA CAGTCGGGGG 6420
  • CTCCCAGCTC TTTGGTTACA AATAGGTTTG GGCCCACAGA GGACGGACCT TGCCCCCTTC 7500 ATGCCTCCCA GGAGACACCT AGCCCCTGCT CTGTGCATGC GGGTGGGCTG GGCCCCCAGG 7560
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO : 9 :
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:27:
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

Cette invention se rapporte à la galactokinase humaine et à l'identification des mutations de la galactokinase, une mutation faux-sens et une mutation non-sens, ainsi qu'à des acides nucléiques isolés codant ces mutations, à une cellule hôte de recombinaison transformée par l'ADN codant ces protéines, et aux utilisations des protéines exprimées et des séquences d'acides nucléiques dans des applications thérapeutiques et diagnostiques.
EP95921460A 1994-09-23 1995-05-26 Gene de la galactokinase humaine Withdrawn EP0783567A4 (fr)

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WOPCT/US94/10825 1994-09-23
PCT/US1995/006743 WO1996009374A1 (fr) 1994-09-23 1995-05-26 Gene de la galactokinase humaine

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* Cited by examiner, † Cited by third party
Title
ELSAS ET AL: "Galactosemia A Molecular Approach to the Enigma" INTERNATIONAL PEDIATRICS, vol. 8, no. 1, 1993, pages 101-109, XP002108635 *
See also references of WO9609374A1 *
STAMBOLIAN ET AL: "Expression and Mutation Analysis of the Gene for Human Galactokinase, a Cause of Congenital and Presenile Cataracts" INVESTIGATIVE OPHTHALMOLOGY AND VISUAL SCIENCE, vol. 36, no. 4, 15 March 1995 (1995-03-15), page S843 XP002109090 *
STAMBOLIAN: "Galactose and Cataract" SURVEY OF OPHTHALMOLOGY, vol. 32, no. 5, 1988, pages 333-349, XP002108634 *

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