WO1996009374A1 - Human galactokinase gene - Google Patents

Human galactokinase gene Download PDF

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Publication number
WO1996009374A1
WO1996009374A1 PCT/US1995/006743 US9506743W WO9609374A1 WO 1996009374 A1 WO1996009374 A1 WO 1996009374A1 US 9506743 W US9506743 W US 9506743W WO 9609374 A1 WO9609374 A1 WO 9609374A1
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WIPO (PCT)
Prior art keywords
seq
galactokinase
sequence
dna
nucleic acid
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PCT/US1995/006743
Other languages
French (fr)
Inventor
Derk Jon Bergsma
Dwight Edward Stambolian
Original Assignee
Smithkline Beecham Corporation
University Of Pennsylvania
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Publication date
Application filed by Smithkline Beecham Corporation, University Of Pennsylvania filed Critical Smithkline Beecham Corporation
Priority to MX9702205A priority Critical patent/MX9702205A/en
Priority to EP95921460A priority patent/EP0783567A4/en
Priority to JP8510858A priority patent/JPH10506529A/en
Priority to AU26536/95A priority patent/AU2653695A/en
Priority to BR9509211A priority patent/BR9509211A/en
Priority to ZA9504371A priority patent/ZA954371B/en
Publication of WO1996009374A1 publication Critical patent/WO1996009374A1/en
Priority to NO971359A priority patent/NO971359L/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
    • C12N9/10—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.
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  • 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

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.

Description

Human Galactokinase Gene
This invention was made in part with government support under EY-09404 awarded by the National Institutes of Health. The U.S. Government has certain rights in the invention.
Cross-Reference to Related Applications:
This application is a continuation in part of Serial No. PCT/US 94/ 10825, filed 23 September 1994.
Field of the Invention:
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.
Background of the Invention: There are numerous inherited human metabolic disorders, most of which are recessive. Many have devastating effects that may include a combination of several clinical features, such as severe mental retardation, impairment of the peripheral nervous system, blindness, hearing deficiency and organomegaly. Most of the disorders are rare. However, the majority of such disorders cannot be treated by drugs.
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)). Patients with homozygous galactokinase deficiency usually become symptomatic in the early infantile period showing galactosemia, galactosura, increased galactitol levels, cataracts and in a few cases, mental retardation (Segal et al., J. Pediatr..9_5_:750-752 (1979)). These symptoms usually improve dramatically with the administration of a galactose free diet. Heterozygotes for galactokinase deficiency are prone to presenile cataracts with the onset during 20-50 years of age (Stambolian et al., Invest. Ophthal. Vis. Sci.. 22:429-433 (1986)).
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.
The first clinical trials of gene therapy began in 1990. Since that time, more than 70 clinical trial protocols have been reviewed and approved by a regulatory authority such as the NLH's Recombinant Advisory Committee (RAC), see, e.g., Anderson, W. F., Human Gene Therapy. 5:281-282 (1994). The therapeutic treatment of diseases and disorders by gene therapy involves the transfer and stable insertion of new genetic information into cells. The correction of a genetic defect by re-introduction of the normal allele of a gene has hence demonstrated that this concept is clinically feasible (see, e.g., Rosenberg et al., New Eng. J. Med.. 323: 570 (1990)). These and additional uses for the reagents described herein will become apparent to those of ordinary skill in the art upon reading this specification.
Summary of the Invention:
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. In yet another aspect, 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
RLFP (for example, by restriction endonuclease digestion of a sample amplified by PCR (for DNA) or PCR-RT (for RNA)). Alternatively, 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).
Brief Description of the Drawings:
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.
Detailed Description of the Invention: 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. In further describing the present invention, 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."
The term "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. For example, 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. For purposes of defining the present invention, 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. Within the promoter sequence will be found 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.
DNA "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. In prokaryotes and yeasts, for example, the exogenous DNA may be maintained on an episomal element, such as a plasmid. With respect to eukaryotic cells, 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" or "transfected" 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.
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.
"Conditions 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. In addition, 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.
Alternatively, "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. As used herein, substantially similar refers to the sequences having similar identity to the sequences of the instant invention. Thus 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. Several particularly useful 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.
This invention also provides for gene therapy. "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. For example, mouse Moloney leukemia virus (MMLV) is a well-known vector in clinical gene therapy trials (see, e.g., Boris-Lauerie et al.. Curr. Opin. Genet. Dev..2:102-109 (1993)).
In contrast, in vivo gene therapy does not require isolation and purification of patients' cells. 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"). 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, fϊbroblasts, lymphocytes, any cell of the eye (e.g., retina), epithelial and endothelial cells. Preferably 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 λ (E. coli . pBR322 (£. coli). pACYC177 (£. coli). pKT230 (gram-negative bacteria), pGVl 106 (gram-negative bacteria), pLAFRl (gram-negative bacteria), pME290 (non-E. £θ_H gram-negative bacteria), pHV14 (£. £pJi and Bacillus subtilis). pBD9 (Bacillus). pIJ61 (Streptomyces). pUC6 (Streptomyces). YIp5 (Saccharornyces). a baculovirus insect cell system, a Drosophila insect system, and YCpl9 (Saccharornyces). See, generally. "DNA Cloning": Vols. I & π, Glover ej - ed. IRL Press Oxford (1985) (1987) and; T. Maniatis s al. ("Molecular Cloning" Cold Spring Harbor Laboratory (1982).
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. 4,431 ,739; 4,425,437; 4,338,397. In addition to control sequences, it may be desirable to add 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). 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. The 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. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
In some cases, it may be desirable to produce other mutants or analogs of the galactokinase protein. 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. 4,446,235; 4,443,539; 4,430,428; ss&alSQ. European Patent Applications 103,409; 100,561; 96,491. 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. are also useful, see for example, PCT applications WO 90/06358 and WO 92/06212 as well as EP 290,261-B1. Depending on the expression system and host selected, 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. The selection of the appropriate growth conditions and recovery methods are within the skill of the art. 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. Schreier ejal., "Hybridoma Techniques" (1980); Hammerling ej si-, "Monoclonal Antibodies and T-cell Hybridomas" (1981); Kennett ej -, "Monoclonal Antibodies" (1980); 5££ al≤Q U.S. Patent Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,452,570; 4,466,917; 4,472,500; 4,491,632; and 4,493,890. Panels of monoclonal antibodies produced against the antigen of interest, or fragment thereof, can be screened for various properties; i.e., for isotype, epitope, affinity, etc. Hence one skilled in the art can produce 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. Alternatively, 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. As used herein, "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. 321:522 (1986); Verhoeyen et al., Science, 239:1534 (1988); Kabat et al., 1 Immunol.. 147:1709 (1991); Queen et al., Proc. Natl Acad. Sci. USA. 86:10029
(1989.: Gorman et al.. Proc. Natl Acad. Sci. USA. 88:34181 (1991); and Hodgson et al., Bio/Technology.9:421 (1991). Therefore, 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).
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. In addition, 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.) is obtained and allowed 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. 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.
This invention also contemplates pharmaceutical 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. Optionally, the galactokinase protein is surrounded by a membrane bound vesicle, such as a liposome.
The 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. A variety of 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. The 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.
Thus, 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. Similarly, 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.
Depending on the patient condition, the pharmaceutical composition of the invention can be administered for prophylactic and/or therapeutic treatments. In therapeutic application, 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. In prophylactic applications, 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. In any event, 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. In addition, one can insert mutant galactokinase genes into a suitable vector for expression in a functional assay system (e.g., colorimetric assay, expression on MacConkey plates, complementation experiments, e.g, in a galactokinase deficient strain of yeast or E. coli) as yet another means to verify or identify galactokinase mutations. As an example, 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. When grown on
MacConkey indicator plates, 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.) In addition, 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. The 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. As an example, 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. In addition, 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)). For example, 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.
Genetic testing based on 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)). In addition, 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)).
Thus, 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. 22Ω: 1242 (1985)) chemical cleavage (e.g., Cotton et al., Proc. Natl. Acad. Sci. USA. £5:4397-4401 (1985))), direct DNA sequencing, or the use of 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. In addition to more conventional gel-electrophoresis, and DNA sequencing, mutations (e.g., microdeletions, aneuploidies, translocations, inversions) can also be detected by 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. See, e.g., Trachuck et al., Science.25Q:559-562 (1990), and Trask et al., Trends. Genet..2: 149-154 (1991) which are incorporated herein by reference for background purposes. Hence, by using nucleic acids based on the structure of specific genes, e.g., galactokinase, one can develop diagnostic tests for galactokinase deficiency.
In addition, some diseases are a result of, or are characterized by, changes in gene expression which can be detected by changes in the mRNA. Alternatively, 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.
See, e.g.. "Current Protocols in Mol. Biol." Vol. I & II, Wiley Interscience. Ausbel £1 al. (ed.) (1992). Probing technology is well known in the art and it is appreciated that the size of the 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. Also within the scope of this invention are 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. Particularly, 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.
Although not necessarily limiting of this invention, following are some experimental data illustrative of this invention.
EXAMPLE I
Purification of Human Galactokinase from Placental Tissue
Galactokinase (galK) 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
[SEQ ID NO:2]
His He Gin Glu His Tyr Gly Gly Thr Ala Thr Phe Tyr Leu Ser Gin- Ala Ala Asp Gly Ala Lys
[SEQ ID NO:3] . Ala Gin Val Cys Gin Gin Ala Glu His Ser Phe Ala Gly Met Pro Cys-
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) were obtained from Human Genome Sciences, Inc. (Rockville, MD, USA). 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). 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. Similarly, SEQ ID NOs: 2 and 3 correspond to amino acids 367-388 and 167-195, respectively, of human galactokinase.
Analysis of the Human Galactokinase Gene:
A comparison of the amino acid sequence for human galactokinase with that of E. coli galactokinase (Debouck et al., Nuc. Acid Res..12:1841-1853 (1985)) shows 61% similarity and 44.5% identity. Further comparison with another purported human galactokinase gene (GK2) (Lee et al., Proc. Natl. Acad. Sci. USA. £2:10887- 10891 (1992)) shows 54% similarity and 34.6% identity at the amino acid level. Furthermore, the GK2 gene maps to human chromosome 15 which is in contrast to the gene of the present invention which maps to human chromosome 17, position q24 as determined by fluorescence in situ hybridization (FISH) analysis.
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.
Galactokinase Complementation:
SEQ ID NO:4 was subcloned into an E. coli vector, plasmid pBluescript [Stratagene]. When transformed into 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. Specifically, 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.
Expression in Mammalian Cells:
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 ,4C 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. For mammalian 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.
Human Galactokinase Gene - Genomic Sequence:
A full-length galactokinase genomic gene coding region was identified from a lambda phage (λ 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
3 May 1995, with the American Type Culture Collection (ATCC), Rockville, MD, USA, under accession number ATCC 97135, and has been accepted as a patent deposit, in accordance with the Budapest Treaty of 1977 governing the deposit of microorganisms for the purposes of patent procedure.
The genomic gene coding region is divided into at least 8 exons isolated from
4 DNA fragments. The arrangement is depicted in Figure 1. The DNA sequence was determined by using multiple oligonucleotide PCR primers corresponding to the galK cDNA sequence (i.e., corresponding to galK genomic exons) as well as oligonucleotide PCR primers subsequently designed that correspond to non-coding regions (i.e., galK genomic introns). Thus the structure of the galactokinase genomic gene is summarized in Table 1 below (see also Figure 2 and SEQ ID NO:7]): Table 1 Genomic Galactokinase Gene
Amino Acids PCR Primer #/
Exon # Encoded [SEQ Lϋ NO]
1 1-55 3333/[8] 3334/[9] 3598/[10] 3599/[l l]
2 56-118 1888/[12] 3332/[13] 3604/[14] 3605/[15]
3 119-158 3331/[16] 3606/[17]
4 159-204 1657/[18] 3034/[19]
5 205-264 3330/[20] 3607/[21]
6 265-315 1539/[22] 2665/[23]
7 316-369 1891/[24] 2665/[25]
8 370-392 2665/[26] 2666/[27] 2667/[28]
Galactokinase Deficiency Marker/Gene:
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. In essence, 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. In contrast, 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.
To determine whether the missense mutation resulted in decreased enzymatic activity, 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. In contrast, 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 Val32 to Met32 substitution as the cause of the decreased enzymatic activity.
Another mutation was discovered in an unrelated patient having cataracts and diagnosed as galactokinase deficient (galactokinase activity was found to be close to zero). Genomic DNA was isolated from lymphoblastoid cell lines and sequenced by automated sequencing on an ABI 373A sequencer. A single base substitution of T for G resulted in an in-frame nonsense codon (i.e., TAG) at amino acid position 80 [SEQ ID NO:6]. This mutation causes premature termination of human galactokinase, resulting in a truncated protein of 79 amino acids that would be expected to be non- functional. (The genomic DNA of the parents of this patient were heterozygous for this mutation, and hence not galactokinase deficient.)
The above description and examples fully disclose the invention including preferred embodiments thereof. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments herein. Such equivalents are intended to be within the scope of the following claims.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Bergsma, Derk J.
Stam olian, D ight
(ii) TITLE OF INVENTION: Human Galactokinase Gene
(iii) NUMBER OF SEQUENCES: 32
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SmithKline Beecham Corp./Corporate Intellectual Property (B) STREET: 709 Swedeland Road/UW2220
(C) CITY: King of Prussia
(D) STATE: Pennsylvania
(E) COUNTRY: USA
(F) ZIP: 19406-0939
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US94/10825
(B) FILING DATE: 23-SEP-1994
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Sutton, Jeffrey A.
(B) REGISTRATION NUMBER: 34,028
(C) REFERENCE/DOCKET NUMBER: P50268-1 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 610-270-5024
(B) TELEFAX: 610-270-5090
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Val Asn Leu lie Gly Glu His Thr Asp Tyr Asn Gin Gly Leu Val Leu 1 5 10 15
Pro Met Ala Leu Glu Leu Met Thr Val Leu Val Gly Ser Pro Arg 20 25 30
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 2 :
His He Gin Glu His Tyr Gly Gly Thr Ala Thr Phe Tyr Leu Ser Gin
1 5 10 15
Ala Ala Asp Gly Ala Lys
20
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ala Gin Val Cys Gin Gin Ala Glu His Ser Phe Ala Gly Met Pro Cys 1 5 10 15
Gly He Met Asp Gin Phe He Ser Leu Met Gly Gin Lys 20 25
(2) INFORMATION FOR SEQ ID NO: :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1349 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA ( ix) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 29..1204
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GAATTCGGCA CGAGTGCAGG CGCGCGTC ATG GCT GCT TTG AGA CAG CCC CAG 52
Met Ala Ala Leu Arg Gin Pro Gin ι 5
GTC GCG GAG CTG CTG GCC GAG GCC CGG CGA GCC TTC CGG GAG GAG TTC 100
Val Ala Glu Leu Leu Ala Glu Ala Arg Arg Ala Phe Arg Glu Glu Phe 10 15 20
GGG GCC GAG CCC GAG CTG GCC GTG TCA GCG CCG GGC CGC GTC AAC CTC 148
Gly Ala Glu Pro Glu Leu Ala Val Ser Ala Pro Gly Arg Val Asn Leu 25 30 35 40
ATC GGG GAA CAC ACG GAC TAC AAC CAG GGC CTG GTG CTG CCT ATG GCT 196
He Gly Glu His Thr Asp Tyr Asn Gin Gly Leu Val Leu Pro Met Ala 45 50 55
CTG GAG CTC ATG ACG GTG CTG GTG GGC AGC CCC CGC AAG GAT GGG CTG 244
Leu Glu Leu Met Thr Val Leu Val Gly Ser Pro Arg Lys Asp Gly Leu 60 65 70
GTG TCT CTC CTC ACC ACC TCT GAG GGT GCC GAT GAG CCC CAG CGG CTG 292
Val Ser Leu Leu Thr Thr Ser Glu Gly Ala Asp Glu Pro Gin Arg Leu 75 80 85
CAG TTT CCA CTG CCC ACA GCC CAG CGC TCG CTG GAG CCT GGG ACT CCT
340
Gin Phe Pro Leu Pro Thr Ala Gin Arg Ser Leu Glu Pro Gly Thr Pro 90 95 100
CGG TGG GCC AAC TAT GTC AAG GGA GTG ATT CAG TAC TAC CCA GCT GCC 388
Arg Trp Ala Asn Tyr Val Lys Gly Val He Gin Tyr Tyr Pro Ala Ala 105 110 115 120
CCC CTC CCT GGC TTC AGT GCA GTG GTG GTC AGC TCA GTG CCC CTG GGG 436
Pro Leu Pro Gly Phe Ser Ala Val Val Val Ser Ser Val Pro Leu Gly 125 130 135
GGT GGC CTG TCC AGC TCA GCA TCC TTG GAA GTG GCC ACG TAC ACC TTC 484
Gly Gly Leu Ser Ser Ser Ala Ser Leu Glu Val Ala Thr Tyr Thr Phe 140 145 150
CTC CAG CAG CTC TGT CCA GAC TCG GGC ACA ATA GCT GCC CGC GCC CAG 532
Leu Gin Gin Leu Cys Pro Asp Ser Gly Thr He Ala Ala Arg Ala Gin 155 160 165
GTG TGT CAG CAG GCC GAG CAC AGC TTC GCA GGG ATG CCC TGT GGC ATC 580
Val Cys Gin Gin Ala Glu His Ser Phe Ala Gly Met Pro Cys Gly He 170 175 180
ATG GAC CAG TTC ATC TCA CTT ATG GGA CAG AAA GGC CAC GCG CTG CTC 628
Met Asp Gin Phe He Ser Leu Met Gly Gin Lys Gly His Ala Leu Leu 185 190 195 200
ATT GAC TGC AGG TCC TTG GAG ACC AGC CTG GTG CCA CTC TCG GAC CCC 676
He Asp Cys Arg Ser Leu Glu Thr Ser Leu Val Pro Leu Ser Asp Pro 205 210 215
AAG CTG GCC GTG CTC ATC ACC AAC TCT AAT GTC CGC CAC TCC CTG GCC
724
Lys Leu Ala Val Leu He Thr Asn Ser Asn Val Arg His Ser Leu Ala 220 225 230
TCC AGC GAG TAC CCT GTG CGG CGG CGC CAA TGT GAA GAA GTG GCC CGG 772
Ser Ser Glu Tyr Pro Val Arg Arg Arg Gin Cys Glu Glu Val Ala Arg 235 240 245
GCG CTG GGC AAG GAA AGC CTC CGG GAG GTA CAA CTG GAA GAG CTA GAG 820
Ala Leu Gly Lys Glu Ser Leu Arg Glu Val Gin Leu Glu Glu Leu Glu 250 255 260
GCT GCC AGG GAC CTG GTG AGC AAA GAG GGC TTC CGG CGG GCC CGG CAC 868
Ala Ala Arg Asp Leu Val Ser Lys Glu Gly Phe Arg Arg Ala Arg His 265 270 275 280
GTG GTG GGG GAG ATT CGG CGC ACG GCC CAG GCA GCG GCC GCC CTG AGA 916
Val Val Gly Glu He Arg Arg Thr Ala Gin Ala Ala Ala Ala Leu Arg 285 290 295
CGT GGC GAC TAC AGA GCC TTT GGC CGC CTC ATG GTG GAG AGC CAC CGC 964
Arg Gly Asp Tyr Arg Ala Phe Gly Arg Leu Met Val Glu Ser His Arg 300 305 310
TCA CTC AGA GAC GAC TAT GAG GTG AGC TGC CCA GAG CTG GAC CAG CTG 1012
Ser Leu Arg Asp Asp Tyr Glu Val Ser Cys Pro Glu Leu Asp Gin Leu 315 320 325
GTG GAG GCT GCG CTT GCT GTG CCT GGG GTT TAT GGC AGC CGC ATG ACG 1060
Val Glu Ala Ala Leu Ala Val Pro Gly Val Tyr Gly Ser Arg Met Thr 330 335 340
GGC GGT GGC TTC GGT GGC TGC ACG GTG ACA CTG CTG GAG GCC TCC GCT
1108
Gly Gly Gly Phe Gly Gly Cys Thr Val Thr Leu Leu Glu Ala Ser Ala 345 350 355 360
GCT CCC CAC GCC ATG CGG CAC ATC CAG GAG CAC TAC GGC GGG ACT GCC 1156
Ala Pro His Ala Met Arg His He Gin Glu His Tyr Gly Gly Thr Ala 365 370 375
ACC TTC TAC CTC TCT CAA GCA GCC GAT GGA GCC AAG GTG CTG TGC TTG 1204
Thr Phe Tyr Leu Ser Gin Ala Ala Asp Gly Ala Lys Val Leu Cys Leu 380 385 390
TGAGGCACCC CCAGGACAGC ACACGGTGAG GGTGCGGGGC CTGCAGGCCA GTCCCACGGC 1264
TCTGTGCCCG GTGCCATCTT CCATATCCGG GTGCTCAATA AACTTGTGCC TCCAATGTGG 1324
AAAAAAAAAA AAAAAAAAAC TCGAG 1349
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1349 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 29..1204 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GAATTCGGCA CGAGTGCAGG CGCGCGTC ATG GCT GCT TTG AGA CAG CCC CAG 52
Met Ala Ala Leu Arg Gin Pro Gin 1 5
GTC GCG GAG CTG CTG GCC GAG GCC CGG CGA GCC TTC CGG GAG GAG TTC 100
Val Ala Glu Leu Leu Ala Glu Ala Arg Arg Ala Phe Arg Glu Glu Phe 10 15 20
GGG GCC GAG CCC GAG CTG GCC ATG TCA GCG CCG GGC CGC GTC AAC CTC 148
Gly Ala Glu Pro Glu Leu Ala Met Ser Ala Pro Gly Arg Val Asn Leu 25 30 35 40
ATC GGG GAA CAC ACG GAC TAC AAC CAG GGC CTG GTG CTG CCT ATG GCT 196
He Gly Glu His Thr Asp Tyr Asn Gin Gly Leu Val Leu Pro Met Ala 45 50 55
CTG GAG CTC ATG ACG GTG CTG GTG GGC AGC CCC CGC AAG GAT GGG CTG 244
Leu Glu Leu Met Thr Val Leu Val Gly Ser Pro Arg Lys Asp Gly Leu 60 65 70
GTG TCT CTC CTC ACC ACC TCT GAG GGT GCC GAT GAG CCC CAG CGG CTG 292
Val Ser Leu Leu Thr Thr Ser Glu Gly Ala Asp Glu Pro Gin Arg Leu 75 80 85
CAG TTT CCA CTG CCC ACA GCC CAG CGC TCG CTG GAG CCT GGG ACT CCT 340
Gin Phe Pro Leu Pro Thr Ala Gin Arg Ser Leu Glu Pro Gly Thr Pro 90 95 100
CGG TGG GCC AAC TAT GTC AAG GGA GTG ATT CAG TAC TAC CCA GCT GCC
388
Arg Trp Ala Asn Tyr Val Lys Gly Val He Gin Tyr Tyr Pro Ala Ala 105 110 115 120
CCC CTC CCT GGC TTC AGT GCA GTG GTG GTC AGC TCA GTG CCC CTG GGG 436
Pro Leu Pro Gly Phe Ser Ala Val Val Val Ser Ser Val Pro Leu Gly 125 130 135
GGT GGC CTG TCC AGC TCA GCA TCC TTG GAA GTG GCC ACG TAC ACC TTC 484
Gly Gly Leu Ser Ser Ser Ala Ser Leu Glu Val Ala Thr Tyr Thr Phe 140 145 150
CTC CAG CAG CTC TGT CCA GAC TCG GGC ACA ATA GCT GCC CGC GCC CAG 532
Leu Gin Gin Leu Cys Pro Asp Ser Gly Thr He Ala Ala Arg Ala Gin 155 160 165
GTG TGT CAG CAG GCC GAG CAC AGC TTC GCA GGG ATG CCC TGT GGC ATC 580
Val Cys Gin Gin Ala Glu His Ser Phe Ala Gly Met Pro Cys Gly He 170 175 180
ATG GAC CAG TTC ATC TCA CTT ATG GGA CAG AAA GGC CAC GCG CTG CTC 628
Met Asp Gin Phe He Ser Leu Met Gly Gin Lys Gly His Ala Leu Leu 185 190 195 200
ATT GAC TGC AGG TCC TTG GAG ACC AGC CTG GTG CCA CTC TCG GAC CCC 676
He Asp Cys Arg Ser Leu Glu Thr Ser Leu Val Pro Leu Ser Asp Pro 205 210 215
AAG CTG GCC GTG CTC ATC ACC AAC TCT AAT GTC CGC CAC TCC CTG GCC 724
Lys Leu Ala Val Leu He Thr Asn Ser Asn Val Arg His Ser Leu Ala 220 225 230
TCC AGC GAG TAC CCT GTG CGG CGG CGC CAA TGT GAA GAA GTG GCC CGG
772
Ser Ser Glu Tyr Pro Val Arg Arg Arg Gin Cys Glu Glu Val Ala Arg 235 240 245
GCG CTG GGC AAG GAA AGC CTC CGG GAG GTA CAA CTG GAA GAG CTA GAG 820
Ala Leu Gly Lys Glu Ser Leu Arg Glu Val Gin Leu Glu Glu Leu Glu 250 255 260
GCT GCC AGG GAC CTG GTG AGC AAA GAG GGC TTC CGG CGG GCC CGG CAC 868
Ala Ala Arg Asp Leu Val Ser Lys Glu Gly Phe Arg Arg Ala Arg His 265 270 275 280
GTG GTG GGG GAG ATT CGG CGC ACG GCC CAG GCA GCG GCC GCC CTG AGA 916
Val Val Gly Glu He Arg Arg Thr Ala Gin Ala Ala Ala Ala Leu Arg 285 290 295
CGT GGC GAC TAC AGA GCC TTT GGC CGC CTC ATG GTG GAG AGC CAC CGC 964
Arg Gly Asp Tyr Arg Ala Phe Gly Arg Leu Met Val Glu Ser His Arg 300 305 310
TCA CTC AGA GAC GAC TAT GAG GTG AGC TGC CCA GAG CTG GAC CAG CTG 1012
Ser Leu Arg Asp Asp Tyr Glu Val Ser Cys Pro Glu Leu Asp Gin Leu 315 320 325
GTG GAG GCT GCG CTT GCT GTG CCT GGG GTT TAT GGC AGC CGC ATG ACG 1060
Val Glu Ala Ala Leu Ala Val Pro Gly Val Tyr Gly Ser Arg Met Thr 330 335 340
GGC GGT GGC TTC GGT GGC TGC ACG GTG ACA CTG CTG GAG GCC TCC GCT 1108
Gly Gly Gly Phe Gly Gly Cys Thr Val Thr Leu Leu Glu Ala Ser Ala 345 350 355 360
GCT CCC CAC GCC ATG CGG CAC ATC CAG GAG CAC TAC GGC GGG ACT GCC
1156
Ala Pro His Ala Met Arg His He Gin Glu His Tyr Gly Gly Thr Ala 365 370 375
ACC TTC TAC CTC TCT CAA GCA GCC GAT GGA GCC AAG GTG CTG TGC TTG 1204
Thr Phe Tyr Leu Ser Gin Ala Ala Asp Gly Ala Lys Val Leu Cys Leu 380 385 390
TGAGGCACCC CCAGGACAGC ACACGGTGAG GGTGCGGGGC CTGCAGGCCA GTCCCACGGC 1264
TCTGTGCCCG GTGCCATCTT CCATATCCGG GTGCTCAATA AACTTGTGCC TCCAATGTGG 1324
AAAAAAAAAA AAAAAAAAAC TCGAG 1349
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1349 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 29..265
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
GAATTCGGCA CGAGTGCAGG CGCGCGTC ATG GCT GCT TTG AGA CAG CCC CAG 52
Met Ala Ala Leu Arg Gin Pro Gin 1 5 GTC GCG GAG CTG CTG GCC GAG GCC CGG CGA GCC TTC CGG GAG GAG TTC 100
Val Ala Glu Leu Leu Ala Glu Ala Arg Arg Ala Phe Arg Glu Glu Phe 10 15 20
GGG GCC GAG CCC GAG CTG GCC GTG TCA GCG CCG GGC CGC GTC AAC CTC 148
Gly Ala Glu Pro Glu Leu Ala Val Ser Ala Pro Gly Arg Val Asn Leu 25 30 35 40
ATC GGG GAA CAC ACG GAC TAC AAC CAG GGC CTG GTG CTG CCT ATG GCT 196
He Gly Glu His Thr Asp Tyr Asn Gin Gly Leu Val Leu Pro Met Ala 45 50 55
CTG GAG CTC ATG ACG GTG CTG GTG GGC AGC CCC CGC AAG GAT GGG CTG 244
Leu Glu Leu Met Thr Val Leu Val Gly Ser Pro Arg Lys Asp Gly Leu 60 65 70
GTG TCT CTC CTC ACC ACC TCT TAGGGTGCCG ATGAGCCCCA GCGGCTGCAG 295
Val Ser Leu Leu Thr Thr Ser 75
TTTCCACTGC CCACAGCCCA GCGCTCGCTG GAGCCTGGGA CTCCTCGGTG GGCCAACTAT 355
GTCAAGGGAG TGATTCAGTA CTACCCAGCT GCCCCCCTCC CTGGCTTCAG TGCAGTGGTG 415
GTCAGCTCAG TGCCCCTGGG GGGTGGCCTG TCCAGCTCAG CATCCTTGGA AGTGGCCACG 475
TACACCTTCC TCCAGCAGCT CTGTCCAGAC TCGGGCACAA TAGCTGCCCG CGCCCAGGTG 535
TGTCAGCAGG CCGAGCACAG CTTCGCAGGG ATGCCCTGTG GCATCATGGA CCAGTTCATC 595 TCACTTATGG GACAGAAAGG CCACGCGCTG CTCATTGACT GCAGGTCCTT GGAGACCAGC 655
CTGGTGCCAC TCTCGGACCC CAAGCTGGCC GTGCTCATCA CCAACTCTAA TGTCCGCCAC 715
TCCCTGGCCT CCAGCGAGTA CCCTGTGCGG CGGCGCCAAT GTGAAGAAGT GGCCCGGGCG 775
CTGGGCAAGG AAAGCCTCCG GGAGGTACAA CTGGAAGAGC TAGAGGCTGC CAGGGACCTG 835
GTGAGCAAAG AGGGCTTCCG GCGGGCCCGG CACGTGGTGG GGGAGATTCG GCGCACGGCC 895
CAGGCAGCGG CCGCCCTGAG ACGTGGCGAC TACAGAGCCT TTGGCCGCCT CATGGTGGAG 955
AGCCACCGCT CACTCAGAGA CGACTATGAG GTGAGCTGCC CAGAGCTGGA CCAGCTGGTG 1015
GAGGCTGCGC TTGCTGTGCC TGGGGTTTAT GGCAGCCGCA TGACGGGCGG TGGCTTCGGT 1075
GGCTGCACGG TGACACTGCT GGAGGCCTCC GCTGCTCCCC ACGCCATGCG GCACATCCAG 1135
GAGCACTACG GCGGGACTGC CACCTTCTAC CTCTCTCAAG CAGCCGATGG AGCCAAGGTG 1195
CTGTGCTTGT GAGGCACCCC CAGGACAGCA CACGGTGAGG GTGCGGGGCC TGCAGGCCAG 1255
TCCCACGGCT CTGTGCCCGG TGCCATCTTC CATATCCGGG TGCTCAATAA ACTTGTGCCT 1315
CCAATGTGGA AAAAAAAAAA AAAAAAAACT CGAG 1349 (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7676 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CCGAGCATCC CGCGCCGACG GGTCTGTGCC GGAGCAGCTG TGCAGAGCTG CAGGCGCGCG 60
TCATGGCTGC TTTGAGACAG CCCCAGGTCG CGGAGCTGCT GGCCGAGGCC CGGCGAGCCT 120
TCCGGGAGGA GTTCGGGGCC GAGCCCGAGC TGGCCGTGTC AGCGCCGGGC CGCGTCAACC 180
TCATCGGGGA ACACACGGAC TACAACCAGG GCCTGGTGCT GCCTATGGTG AGGGGCTGCA 240
CGGGGAGCCC CTAGCCCGCC GCCGCCTGTC CCGGTCGCCG AGGAGGGCGG GCCTCGGGGA 300
CGCTGGGGGC GAGTTCTTCC CGCGGGAGAT GTGGGGCGGG CAGCTGCGCC TGGAGCACCG 360
GTGCACGGAA GAGTCCCCGG GACAGGCTGT TCCCCACGTT GGAAGGGAGG AAGCGAAGAA 420
GTGGTCCCCA GAGGGTGCGC GGCCGCCTCT TGGCTCAAGC CCGCCCTCTG GGGGCTGGGG 480 CTCCTCGCCT TCAACCTGGG AGCATGTTCC CCTTAAACTG TGAGGCCCTG TGTGCCACGC 540
AGAAGGGGAC ACTCCGCGCC TCCGGCCACC GTGGGGCCCC AACCGCAGAC CTGGGCGAAC 600
GTAGCCTTCT GGCCCAGCCC GTTCAATTTA CAGAGGAGGA AACTGAGGCC TAGAGAGGCC 660
CAGTGAACTG CTGGAGGTCA CACAGCAGGT TCTTGGCGGG GCTGCGACTT GGGAGTGAGG 720
ACTCCCAGCT TTCAGCGGGG GGCGCTTTCC GCCCCATCTG CAGCTTGGGG AGTGCACAGG 780
TACAGGATGT CCAGAGCCAC CCAAAATGTA AAGGCTTTGG AGCTCCAGTG ATCTGTTTTC 840
CCTTTGGGCT AAGCTCTCCC CCCTTGCCCC ACAGCTCAGG GCAGAGTCCA GGTCTGTGCT 900
CCAGCTGCAG CCGCCCCGCC CCTGAAGACC TAAGGGGGCA GGGCTCAAGC CCCCAAGGTC 960
AGCTGGCCCT CAGGATCTTC CCTGCGACGC TGAACCTGGA GGTTCAGAAC CTGATGACTG 1020
TGGAGGCATC AGAACCTCGG CTGGAGGCAG TGTCATTGGA GAGGCTTACT CCAGCTGGCG 1080
GAAGCCTCAC GTACTGCTTG TCTCTCCTGC CAGGCTCTGG AGCTCATGAC GGTGCTGGTG 1140
GGCAGCCCCC GCAAGGATGG GCTGGTGTCT CTCCTCACCA CCTCTGAGGG TGCCGATGAG 1200
CCCCAGCGGC TGCAGTTTCC ACTGCCCACA GCCCAGCGCT CGCTGGAGCC TGGGACTCCT 1260 CGGTGGGCCA ACTATGTCAA GGGAGTGATT CAGTACTACC CAGGTATGGG GCCCAGGCCT 1320
GAGCCAAGTC CTCACTGATA CTAGGAGTGC CACCTCACAG CCACAGAGCC CATTCATTTG 1380
TCTGATACAC TGTGGGGAAG GCTTGTAGAG TGGAGCATCC CATTGTACAG ATGAGGAAAC 1440
TGATGCCCCC AGAAGGTCGG GAACTTGCCC TGGGTTTCCC GTGACCTGAT TGGAGGAGCC 1500
AGGATTTGAA CCCCAGCCTT TTTTCCCTCC AGAGCCCTAA ACCAGGAGGA CAATTAGAAG 1560
TGTCCCAGCA ACCTCAGAGG GTGGGAAAAT GGAGGGGAGT GGGTCCCTTG GGCCAGCAGG 1620
TTGGTGGGGT TCTTGACAAT TGAGACACAC ACCTAGAAAC AGTTGCTAGG CCGTTGCTGC 1680
CCTTCCCGCC AGGACACCTG CCCTTCCTGT CCAATCCTCC CAGGCAGCCT CTCTTACCAT 1740
CACCTGTTCT TTCCCCCTGC AGCTGCCCCC CTCCCTGGCT TCAGTGCAGT GGTGGTCAGC 1800
TCAGTGCCCC TGGGGGGTGG CCTGTCCAGC TCAGCATCCT TGGAAGTGGC CACGTACACC 1860
TTCCTCCAGC AGCTCTGTCC AGGTACCAGC TAGGCCCCAG CCCTGACCCA GCCCTCCTTC 1920
CCTGAGGTCT CCAGGTGGTC CCAGCTTCTA CTATGCCTTA TGGAGGGGGT GGCAGGGAAT 1980
CTCCCTGGAG TGTCATTGAA GCCACTGCTG CTTCCACCAG CCCTAGCCTC CCCACCTCAC 2040 CCTGTACTGC AGACTCGGGC ACAATAGCTG CCCGCGCCCA GGTGTGTCAG CAGGCCGAGC 2100
ACAGCTTCGC AGGGATGCCC TGTGGCATCA TGGACCAGTT CATCTCACTT ATGGGACAGA 2160
AAGGCCACGC GCTGCTCATT GACTGCAGGT TGGGCTCGCT CCCCTCGTCC CCTCCCGCCC 2220
TGCACTCAGC AGCTCCTGGG TGGAGTGTGC CCACTGCCTG GCGCAGCAAG CACACGCTTG 2280
GCCTCGTCAT CTCCCCCATT GTAACTCCAC CCCAGGTCCT TGGAGACCAG CCTGGTGCCA 2340
CTCTCGGACC CCAAGCTGGC CGTGCTCATC ACCAACTCTA ATGTCCGCCA CTCCCTGGCC 2400
TCCAGCGAGT ACCCTGTGCG GCGGCGCCAA TGTGAAGAAG TGGCCCGGGC GCTGGGCAAG 2460
GAAAGCCTCC GGGAGGTACA ACTGGAAGAG CTAGAGGGTG AGAACTGCCA GGGTGCTCTA 2520
TCCTGGAGGC GGCTGTGCTC CCTGCTGGCG CCTCAGTGTG GCCTTGACCC TGCCTGGGAC 2580
CCCGATCTCC AGGGGCTTCT GCCATGCTCT CCCCAGTCCC TTCAAACACT GCGCACCCAG 2640
GGTTCCAATC TCAGCAGGGG TGCTTGAAAT CCTAAAATGG TCTTATCTAA TCAGAAAAAT 2700
CATGTTTCCA TTGTGGAAAA TGTAGAAAAG TACAAAGTAG AAAATAATAA GCTATAAGGG 2760
CACTACCCAG AGATAGGCAC TGCTGACATT TTCACGTTTC CTTTCAGTAT TTTTCCACAT 2820 CTGTCTTCAA AGCTGAGTAT ATGTAATATA TCATCACTTT CCCCCCCCAC CCCCTTTTTT 2880
TTAAGAGGCA GGGTCTCATT CTGTTGCCCA AGCTGGAGTG TAGTGGTGTG ATCATAGCTT 2940
ACTGCAAACT TGAACTCTTG AGCTCAAGGG ATCCTCCCAG CTCAGCCTTC CAAGTAGCTG 3000
AGATTACAGG TGTGCCACCA TGCCCGGCTA ATTTTTATCT TCGTAAAGAC GGCCTTGTAG 3060
TGTTGCCCAG GATGATCCTG AACTCTGGCC TCAAGAGGTC CTCCTGCCTT GGGCTCCCAA 3120
AGTGTTGGGA TTATAGGCAT GAGCCACTGC GGCCAGCCCA TTTGCCGTGT TTTTTTTTTG 3180
GACACAGAGT TTCGGTCTTG TCACCCATGC TGGAGTGCAA TGGTGCGATC TCAGCTCACT 3240
GTAACCTCTG CCTCCCGGGT TCAAGTGATT CTCCTGCCTC AGCCTCCCGA GTAGCTGGGA 3300
CTACAGGCGC CCGCCACTAC GCCTGGCACA TTTTTTATAG TTCTAGTAGA GACTGGGGTT 3360
TCACCATGTT GGCCAGGCTG GTCTCAAACG CCTGACCTCA GGTGATCCTC CCGCCTCAGC 3420
CTTCCAAAGT GCTGGGATTA CAGGCGTGAG CCATAGTGCC GGTCTCTTTT TTTTTTTTTT 3480
TTAAACTAAA CATAATCTCA GAACCCAGAA CCCTATCTTA TCTTATGCCA TGAAAGGCAT 3540
ATCTCGGCGT GGCTCTTTTT TTTTTTTTTT CTTTTTTTTT GGGCGAGGTG GAGGCTTGCC 3600 CTGTTGCCCA GGCTGGAGTG CAGCGGCGCA ATCTCGGTTC ACTGCATCCT CCACCTCCTG 3660
GGTCCAAATG ATCCTCCTGC CTTAGCTTCC TGAGTAGGTG GGATTACTGG AACCCACCAC 3720
CACGCCCAGC CAATTTTTAT ATTTTTAGTA GAGACGGGGT TTCATGTTGG CCAGGCTGGC 3780
CTCGAACTCC TGACCTCGTG ATCTGCCCGC CTCAGCCTCC CAATGTGCTA GGATTACATG 3840
TGTGAGCCAC TGCACCTGGC CTCCGTGTGG CTCTTTAAAG CTCCACAATA TTTTAGCATT 3900
CAGGTGCTCT GTCATTTACT TAACTATTTT CTGATACACC TCACACTGCG ATTAACTTTC 3960
CTTATTTATC TTTTTTATTA TTTATTTATT TATTTATTTG AGACAGAGTC TTGCTCTGTC 4020
ACCCAGGCTG GAGTGCAGTG GCACGATCTC GGCTCACTGC AACCTCTGCC TCCCAGGTTC 4080
AAGTGATTCT CCTGCCTCAG CCTCCTGAGT AGCTAGGATT AGAGGCATGT GCCACCACAC 4140
CTGGCTAATC TTCGTATTTT TAGCAGAGAT GAGGTTTTAC CATGTTGGTC GGGCTGGTCG 4200
TGAACTCCTG ACCTGGTGAT CTGCCCACCT CAGCCTCCCA AAGTACTGGG ATGACAGGCA 4260
TGAACCACTG TGCCTGGCCA TCTTTTTTAT TTTTTAAAGA GATGGGTTCT GCTAAGTTGC 4320
CCAGGCTGGA CCTGAACTCT TGGGCTCAAG TAATCTTCTC ACCTAGTCTC CTGGGTAGCT 4380 GCAACCAAAG GCACCCGGTT TATCTGCATT CTCTTTTTTT TCTTTGAGAC TGAGTCTTGC 4440
TCTGTAGCCC AGGCTGGAGC GCAGTGGCGT GATCTCGGCT CACTGCAACC TCCGTCTTCA 4500
GGGTTCAAGC AATTCTCCTG CCTCAGCCTC TGGAGTGGCT GGGACTACAG GCGTGTGCCA 4560
CCAGAGCGAG TTAATTTTTT TTTTTTTTTG TATTTTTAGT GGACACTGGG TTTCACTATA 4620
TTGGCCAGGC TGGTCTTGGA CTCCTGACCT CAAGTGATCC GCCTGCCTTG GCCTCCCAAA 4680
GTGCTGGGAT TACAGGCACA GGCGTGAGCC ACTACACCTG GCCTATCTGC ATTCTCTTAA 4740
TAGTTTCTTA GAAATGGATT CTTAGGAGTA GGATTACAGA GTCAAGAGAC ACAAGTTTTG 4800
TAGGCTGGGT GCGGTGGCTC ACGTCTGTGC CTGTAATCCC AGTACTTTAG GAGGCCAAGG 4860
TGGGCAGATT CATTGAGCTC AGGAATTCGA GACCAGCCTG GGCAACATGG CAAAACCCCA 4920
TCTCTAAAGA AATACAAAAA TTAGCCAGGT GTGGTGGTGT GTGCCTGTAG TCCTAGCTAC 4980
TTAGGAGGCT GGGGTGGGAG GATCAATTGA GCCCAGGAGG TTGAGACTGC AGTGAGCTGT 5040
GATTGCACCA TGGCACTCCA GCCTGGGCCT CAAAGTGAGA TCCTGTCTCC AAAACAAAAA 5100
AGATACAAGT ATCCTTAAGG CTCCTGCTAC ACATGGCCAG GAAGGTAGTC TATTGGACAG 5160 TTTTAAGGTC ATTATCAATA TTAGCTCATT TAATTCCCTC CAAAACTCTG TAAAGCACAT 5220
TCTGCTACCA TAGTTGTCAT ATTTTTGATG GGGGAATCTA CAGTGAGAGG CAGTGCTGGG 5280
ATCTGAACCC CATCTGGACA GATTAGCTCC AGGGCCCATG CTCTTGACTG GCTGGCCGCG 5340
CTGCCCACAC TGAGTTGTTC CTTCCTGGCA GGGTAGGTGT GCCTATCTCA GGGACACTAG 5400
ACAGCTCCGA GGGACCTCCC TGTCCTTTTC CTTTGTGAAC TGTGTCACGT TCTCCAGAGC 5460
AGGGCTCAGA CCTGCCCTGC CTGCTCTGTG CAGATGCCCT TGGCCAAGGT TTTCACACTG 5520
GAACAAGTTG GTCCCTCCTC CCCACCCCAG CCTGTCCTTG GCCCTCCTCC AGGTCTCCTT 5580
CTGCATAGGA GCAGCTCACC CTGCCTCCTC CAGAGTCCTG CCCTAGAAGC GCAATCCCTC 5640
TCCTTCCATC CCCTGCCTGG CTGCCTGGCT CCTTCCCTCA GCCTCCAAGA CATGCTCAGT 5700
TTTCTTCCCT CCTAAAACAC CACCCACTGT CTCATTTCCA TTCATTTCTT TCTTTCTTTC 5760
TTTCTTTTTT TTTTTTGAGA GGGAGCCTCA CTCTGTCACC CAGGCTGAAG TGCAGTGGCA
5820
TGATCTCCAC TCACTGCAAC CTCCGCCTCC CAGGTTCAAG CAATTCTCCT GCCTCAGCCT 5880
CCTGAGTAGC TGGGATTACA GGCGCCTGCC ACGATGCCCG GCTAACTTTT GTATTTTTAG 5940 TAGAGACGGG GTTTCGCCAT GTTGGCCAGG CTGGTCTCGA GCTCCTGACC TCAGGCAATC 6000
TGCCTGCCTC AGCTTCCCAA AGTGCTGGGA TTACAGGTGT GAGCCACCGC GCCCACCCAT 6060
TCATTTCTCA GTCCTTTGAA TCTACTTGCC CCTCCATCCC GCCATGCCAC CTACCCTAAC 6120
AACCTTCCCC CTTAAACCTG CGGGTTTGGC CGGGCGCAGT ACACTGAGTC AGTACTGGTA 6180
CTGACCCAGG TACCCCTCCA GCCTCAGCTC CAGTCAGATG GGACAGCCTG CTGGTCCCTG 6240
GCTGCTTCTG CCCCCTCTTC TGGAGCCCCA GCCCTGGAGG CTCCATGTGG CTCAGCAGAA 6300
CTTCTTCTCC TCCTGCTCTG TGGTGGCCTC TTGAGGGCAG CACTCACCTT GGAAAGCATG 6360
GAGTGTTTCA ACCCTCACTG CTCCCTGAAG GACCAAGGTG TCCCATTTTA CAGTCGGGGG 6420
AGGAGGCACT GTGATAAAGG GGCTCTTCAG ACCCACGTCT GAGAGAGCCA GGCTGCGCCG 6480
CCCCCGCGGC CTTCCACCCT TCACCGTCCA GCCAGGGCCA CTGCCATCAC CGCCTGCTGG 6540
TCCTCACAGG CGTCGGGGCC CCAGGCAGTG AGAAGGCGGC TGCTGACTCC TCTTTCCTCC 6600
CCAGCTGCCA GGGACCTGGT GAGCAAAGAG GGCTTCCGGC GGGCCCGGCA CGTGGTGGGG 6660
GAGATTCGGC GCACGGCCCA GGCAGCGGCC GCCCTGAGAC GTGGCGACTA CAGAGCCTTT 6720 GGCCGCCTCA TGGTGGAGAG CCACCGCTCA CTCAGGTGAG GCCCTCTGGG CGCCCCGCTC 6780
CTGCCGGGCA CAGGCCGGCC CAGGCCCACC CCTTCAATAT CCTCTCTGCA GAGACGACTA 6840
TGAGGTGAGC TGCCCAGAGC TGGACCAGCT GGTGGAGGCT GCGCTTGCTG TGCCTGGGGT 6900
TTATGGCAGC CGCATGACGG GCGGTGGCTT CGGTGGCTGC ACGGTGACAC TGCTGGAGGC 6960
CTCCGCTGCT CCCCACGCCA TGCGGCACAT CCAGGTGGGC GGGCACCAGG GCCTGGGCGG 7020
GCAGGAGCGG CAGCTTCCCG GGGCCCTGCC ACTCACCCCC AGCCCGCCTC TTACAGGAGC 7080
ACTACGGCGG GACTGCCACC TTCTACCTCT CTCAAGCAGC CGATGGAGCC AAGGTGCTGT 7140
GCTTGTGAGG CACCCCCAGG ACAGCACACG GTGAGGGTGC GGGGCCTGCA GGCCAGTCCC 7200
ACGGCTCTGT GCCCGGTGCC ATCTTCCATA TCCGGGTGCT CAATAAACTT GTGCCTCCAA 7260
TGTGGTACCT GCCTCCTCTA GAGGTGGGTG TATGCTTGGG TGTCAGAGAA TGGGGGATGT 7320
CAGAACCGCT CCCCTACCCT AGGGGAGCAC CTCTCAGGCC CCAGAAGAAT GGGCAAGGCA 7380
GGGCCTAGCA GTAGCAAAAC CATTTATTAA GTGCAGAACA AAGGCTGGGT CCTTGTGCTG 7440
CTCCCAGCTC TTTGGTTACA AATAGGTTTG GGCCCACAGA GGACGGACCT TGCCCCCTTC 7500 ATGCCTCCCA GGAGACACCT AGCCCCTGCT CTGTGCATGC GGGTGGGCTG GGCCCCCAGG 7560
GGTGCAAGGA TGGAGTAGCT GAGGAGGCTC CGGGAGAGGA GTCGGGAGGA CGCCTAGTGG 7620
GACATTGCGG GGGTGGCGCA GGGTGCGGTC AAGTTTGGAA GAAACTGTTG GGTCCA 7676
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
AGCCTTCCGG GAGGAGTTCG G 21
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 9 :
CTGGTTGTAG TCCGTGTGTT C
21
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GCCAGCAGCT CCGCGACCTG G 21
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
GCTTCCTCCC TTCCAACGTG G 21 (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
CCCAGGCTCC AGCGAGCGCT G 21
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
ACCTCTGAGG GTGCCGATGA G 21
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH : 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
CCCACAGCTC AGGGCAGAGT C 21
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
GGACACTTCT AATTGTCCTC C 21
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GATGAACTGG TCCATGATGC C 21
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
AGGGGCACTG AGCTGACCAC C 21
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) ( i) SEQUENCE DESCRIPTION: SEQ ID NO:18:
CACTTCTACA CATTGGCGCC G 21
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
CTTCGCAGGG ATGCCCTGTG G 21
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
( i) SEQUENCE DESCRIPTION: SEQ ID NO:20: TCATCACCAA CTCTAATGTC C 21
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21
TGTCAGCAGT GCCTATCTCT G 21
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22
AGCAGCGGAG GCCTCCAGCA G
21
(2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CCTCACCGTG TGCTGTCCTG G 21
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
GGCTGCGCTT GCTGTGCCTG G 21
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
CCTCACCGTG TGCTGTCCTG G 21
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
CCTCACCGTG TGCTGTCCTG G 21
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
GCGGGACTGC CACCTTCTAC C 21
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
CTCAATAAAC TTGTGCCTCC A 2i
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
CGGATATGGA AGATGGCACC GGG 23
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
AGAGCTGCAG GCGCGCGTCA TG 22
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
CCGAGCATCC CGCGCCGAC 19 (2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
( i) SEQUENCE DESCRIPTION: SEQ ID NO:32:
CAGCTGCCCG CCCCACATCT 20

Claims

WHAT IS CLAIMED IS:
1. An isolated nucleic acid molecule encoding human genomic galactokinase, said nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising the sequence as set forth in SEQ ID
NO:7; and
(b) a nucleic acid molecule differing from the nucleic acid molecule of (a) in codon sequence due to the degeneracy of the genetic code.
2. A vector comprising the nucleic acid molecule of claim 1.
3. A recombinant host cell comprising the vector of claim 2.
4. An isolated nucleic acid molecule comprising a DNA sequence that encodes nucleotides 29 to 1204 of SEQ ID NO:5 or nucleotides 29 to 265 of SEQ ID NO:6.
5. A vector comprising the nucleic acid molecule of claim 4.
6. The vector according to claim 5 which is a plasmid.
7. A recombinant host cell comprising the vector of claim 5.
8. A process for preparing a human galactokinase protein comprising culturing the recombinant host cell of claim 7 under conditions promoting expression of said protein and recovery thereof.
9. An isolated protein encoded by the DNA sequence of claim 4.
10. A monoclonal antibody that is specifically reactive with the protein of claim 9.
11. A method for diagnosing conditions associated with human galactokinase deficiency which comprises isolating a serum or tissue sample from an individual; allowing such sample to come in contact with an antibody or antibody fragment which specifically binds to the human galactokinase protein of claim 9 under conditions such that an antigen-antibody complex is formed between said antibody or antibody fragment and said galactokinase protein; and detecting the presence or absence of said complex.
12. A method for diagnosing conditions associated with human galactokinase deficiency which comprises isolating a nucleic acid sample from an individual; assaying said sample and the DNA sequence, or corresponding RNA sequence, that encodes a human galactokinase gene; and comparing differences between said sample and said DNA (or RNA) that encodes nucleotides 29 to 1204 of SEQ ID NO:4, wherein said differences indicate mutations in the human galactokinase gene.
13. The method of claim 12 wherein said sample is RNA which is subsequently amplified by PCR-RT.
14. The method of claim 13 wherein assaying said sample comprises a restriction endonuclease digestion.
15. The method of claim 14 wherein said restriction endonuclease is Msc I.
16. The method of claim 12 wherein assaying said sample comprises a hybridization assay.
17. The method of claim 16 wherein the hybridization assay is heteroduplex electrophoresis which comprises determining differential mobility of heteroduplex products in polyacrylamide gels, said heteroduplex products are the result of hybridization between the nucleic acid sample and the DNA sequence, or corresponding RNA sequence, that encodes nucleotides 29 to 1204 of SEQ ID NO:4.
18. The method of claim 12 wherein assaying said sample comprises gel electrophoresis of restriction fragment length polymorphisms of said nucleic acid sample and the DNA sequence, or corresponding RNA sequence, that encodes nucleotides 29 to 1204 of SEQ ID NO:4.
19. The method of claim 12 wherein assaying said sample comprises DNA sequencing.
20. A method for diagnosing conditions associated with human galactokinase deficiency which comprises isolating cells from an individual containing genomic DNA and assaying said sample by in situ hybridization using the DNA sequence that encodes nucleotides 29 to 1204 of SEQ ID NO:4, nucleotides 29 to 1204 of SEQ LD NO:5, or nucleotides 29 to 265 of SEQ ID NO:6; or a fragment that encodes at least one exon of said sequence; or a fragment containing at least 15 contiguous base pairs of said sequence as a probe.
21. A transgenic non-human mammal capable of expresing in any cell thereof the DNA of claim 4.
PCT/US1995/006743 1994-09-23 1995-05-26 Human galactokinase gene WO1996009374A1 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
MX9702205A MX9702205A (en) 1994-09-23 1995-05-26 Human galactokinase gene.
EP95921460A EP0783567A4 (en) 1994-09-23 1995-05-26 Human galactokinase gene
JP8510858A JPH10506529A (en) 1994-09-23 1995-05-26 Human galactokinase gene
AU26536/95A AU2653695A (en) 1994-09-23 1995-05-26 Human galactokinase gene
BR9509211A BR9509211A (en) 1994-09-23 1995-05-26 Human galactokinase gene
ZA9504371A ZA954371B (en) 1995-05-26 1995-05-29 Human galactokinase gene.
NO971359A NO971359L (en) 1994-09-23 1997-03-21 Human galactokinase gene

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
USPCT/US94/10825 1994-09-23
PCT/US1994/010825 WO1996009408A1 (en) 1994-09-23 1994-09-23 Human galactokinase gene

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JP (2) JPH10505757A (en)
AU (2) AU7958194A (en)
BR (1) BR9509211A (en)
MX (1) MXPA94008258A (en)
NO (2) NO971359L (en)
PL (1) PL319344A1 (en)
WO (2) WO1996009408A1 (en)
ZA (1) ZA947865B (en)

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WO2000009705A2 (en) * 1998-08-11 2000-02-24 Danisco A/S Selection method

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CN1324941A (en) * 2000-05-19 2001-12-05 上海博德基因开发有限公司 New polypeptide human galactokinase 11 and polynucleotides for encoding same

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GB9016984D0 (en) * 1990-08-02 1990-09-19 Ici Plc Aniline derivatives

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BIOCHIMICA BIOPHYSICA ACTA, Volume 831, issued 1985, D. STAMBOLIAN et al., "Purification of Human Galactokinase and Evidence for its Existence as a Monomer Form", pages 306-312. *
J. BACTERIOL., Volume 172, No. 8, issued August 1990, H.H. HOUNG et al., "Molecular Cloning and Physical and Functional Characterization of the Salmonella Typhimurium and Salmonella Typhi Galactose Utiltization Operons", pages 4392-4398. *
MOL. MICROBIOL., Volume 10, No. 2, issued 1993, P. GLASER et al., "Bacillus Subtilis Genome Project: Cloning and Sequencing of the 97 kb Region from 325 Degrees to 333 Degrees", pages 371-384. *
NUCL. ACIDS RES., Volume 13, No. 6, issued 1985, C. DEBOUCK et al., "Structure of the Galactokinase Gene of Escherichia Coli, the Last (?) Gene of the Gal Operon", pages 1841-1853. *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000009705A2 (en) * 1998-08-11 2000-02-24 Danisco A/S Selection method
WO2000009705A3 (en) * 1998-08-11 2000-06-15 Danisco Selection method
US6924145B1 (en) 1998-08-11 2005-08-02 Danisco A/S Selection method

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JPH10505757A (en) 1998-06-09
JPH10506529A (en) 1998-06-30
NO971363D0 (en) 1997-03-21
EP0783567A1 (en) 1997-07-16
BR9509211A (en) 1998-01-27
NO971359D0 (en) 1997-03-21
ZA947865B (en) 1995-10-09
EP0783567A4 (en) 1999-09-15
WO1996009408A1 (en) 1996-03-28
PL319344A1 (en) 1997-08-04
NO971359L (en) 1997-05-21
MXPA94008258A (en) 2005-08-26
AU7958194A (en) 1996-04-09
AU2653695A (en) 1996-04-09

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