WO2000037617A1 - Glutamine: une enzyme de type ii, la fructose-6-phosphate aminotransferase, et ses acides nucleiques codants - Google Patents

Glutamine: une enzyme de type ii, la fructose-6-phosphate aminotransferase, et ses acides nucleiques codants Download PDF

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Publication number
WO2000037617A1
WO2000037617A1 PCT/US1999/030943 US9930943W WO0037617A1 WO 2000037617 A1 WO2000037617 A1 WO 2000037617A1 US 9930943 W US9930943 W US 9930943W WO 0037617 A1 WO0037617 A1 WO 0037617A1
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nucleic acid
gfat
acid molecule
protein
cell
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PCT/US1999/030943
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English (en)
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Marvin Coughenour
Yumi Kasai
Stanton Baldwin Dotson
Mark Gerald Currie
Kay O. Broschat
William M. Moore
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G.D. Searle & Co.
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Priority to AU22163/00A priority Critical patent/AU2216300A/en
Publication of WO2000037617A1 publication Critical patent/WO2000037617A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)

Definitions

  • GLUTAMINE FRUCTOSE-6-PHOSPHATE AMINOTRANSFERASE II ENZYME AND THEIR ENCODING NUCLEIC ACIDS
  • the present invention relates to a mammalian cDNA encoding a glutamine: fructose-6-phosphate amidotransferase-II (GFAT II) protein.
  • the invention also relates to nucleic acid molecules associated with or derived from this cDNA including the complements, homologues and fragments thereof, and methods of using these 10 nucleic acid molecules, to generate, for example, enzymes and fragments thereof.
  • GFAT fructose-6-phosphate amidotransferase
  • amidotransferase domain the NTN- and triad classes (Zalkin, In: Advances in
  • NTN- amidotransferases are distinguished from other amidotransferases by an N-terminal cysteine, which is a participant in the catalytic transfer of amide to an acceptor molecule.
  • the active site includes an internal cysteine and forms
  • GFAT is the only amidotransferase that is unable to utilize free ammonia.
  • the other two mammalian NTN family members are Asparagine synthetase (Asn Syn) and PRPP amidotransferase.
  • GFAT consists of a glutaminase domain (26.4 kDa), which catalyzes hydrolysis of glutamine to glutamic acid and ammonia, and a synthase domain (40.5 kDa), which catalyzes amination of fructose-6-phosphate (Zalkin, In: Advances in Enzymology, Vol. 66, pp.
  • glucosamine-6-phosphate Metabolism of the glucosamine-6-phosphate leads to the formation of UDP-N- acetylglucosamine, which is the precursor of glycoproteins and proteoglycans.
  • Levels of free glucosamine-6-phosphate are very low in tissues, because the majority of glucosamine is acetylated and bound to UDP (UDP-GlcNAc) or covalently bound to macromolecules.
  • UDP-GlcNAc UDP
  • Products of the hexosamine pathway have been postulated to serve a second function, i.e. accumulation of hexosamines that result in the induction of insulin resistance (Marshall et al, FASEB J. 5:3031-3036 (1991); McClain and Crook, Diabetes 45:1003-1009 (1996); both of which are herein incorporated in their entirety).
  • Non-insulin dependent diabetes mellitus so-called "type 2" diabetes, involves insulin resistance (reduced action of insulin at target tissues) which leads eventually to the failure of the pancreatic ⁇ cells to synthesize enough insulin to overcome the insulin resistance. The result is fasting hyperglycemia.
  • Insulin resistance can also be induced by incubating adipocytes and skeletal muscle with glucosamine (in place of glucose) (Marshall et al., FASEB J. 5:3031- 3036 (1991); Thompson et al., J. Biol. Chem. 272:7759-7764 (1993); Robinson et al., Diabetes 42:1333-1346 (1993); the entirety of which is herein incorporated by reference).
  • Glucosamine is more potent than glucose at inducing insulin resistance.
  • Skeletal muscle is one of the tissues that displays insulin resistance.
  • GFAT activity in skeletal muscle biopsy samples from NIDDM patients was measured and compared to age/weight-matched controls, GFAT was found to be elevated in muscle from NIDDM patients (Yki-Jarvinen et al, Diabetes 45:302-307 (1996); the entirety of which is herein incorporated by reference). GFAT activity in muscle has
  • the present invention is directed, in part, to the isolation of a GFAT II protein- encoding nucleic acid, which exhibits extensive sequence homology to GFAT.
  • GFAT II and GFAT exhibit different expression profiles, suggesting additional or different roles for GFAT II in cellular metabolism and in an organism as a whole.
  • specific inhibitors of GFAT II will have fewer side effects and greater therapeutic potential in treating insulin resistance and diabetes.
  • the potential for reducing insulin resistance may be more promising by approaching GFAT II specifically, or, alternatively, by considering effects on both GFAT II and GFAT.
  • the present invention comprising novel GFAT II nucleic acids, proteins, peptides, fragments, and homologues, provides, inter alia, new and advantageous targets to screen for diagnostic and therapeutic agents and compositions, for example, those useful to diagnose or treat NIDDM and related diseases.
  • the invention provides a substantially pure nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 3, 5, 7, or complements thereof; nucleic acid sequences that specifically hybridize to SEQ ID NO: 3, 5, 7, or complements thereof, espescially those that hybridize under stringent conditions; nucleic acid sequences encoding a GFAT II protein or fragment thereof, or complement of either; and nucleic acid sequences encoding the amino acid sequence of SEQ ID NO: 4, 6, 8, or complements thereof.
  • the invention also provides a substantially pure fragment GFAT II nucleic acid molecule, which comprises a nucleic acid sequence that is identical to at least about 19 contiguous nucleotides of SEQ ID NO: 3, 5, 7, or their complements, or at least about 50 or about 100 contiguous nucleotides. Additionally, GFAT II nucleic acid molecules and fragment GFAT II nucleic acids molecules can possess about 70% to about 95% sequence identity over a region of about 20, or about 50 to about 100 contiguous nucleotides of SEQ ID NO: 3, 5, 7, or their complements.
  • the invention also provides a substantially pure murine, human, or rat GFAT II nucleic acid molecule, which comprises a nucleic acid sequence that is identical to at least about 19 contiguous, or about 50 to about 100 contiguous nucleotides of SEQ ID NO: 3, 5, or 7, or their complement. Homologues and polymorphic sequences, especially single polymorphic sequences, of the murine, human, and rat nucleic acids of SEQ ID NO: 3, 5, and 7 are also provided. Thus, nucleic acid molecules of the invention may be used to obtain any mammalian GFAT II homologue.
  • a subset of the nucleic acid molecules of the invention includes hybridization or PCR probes, which can be used, for example, to identify mammalian GFAT II homologue nucleic acids and genes. These probes can also be used to identify genomic clones of GFAT II, especially mouse, human, or rat genomic clones, or to identify genomic regions flanking the GFAT II gene.
  • a nucleic acid of the invention will hybridize to a GFAT II sequence, such as SEQ ID NO: 3, 5, or 7, or complements thereof, or about 20 to about 50 or about 100 contiguous nucleotides of SEQ ID NO: 3, 5, or 7, or complements of them, but will not hybridize to a GFAT sequence, such as SEQ ID NO: 1, or complement thereof, or about 20 to about 50, or to about 100 contiguous nucleotide fragments of SEQ ID NO: 1.
  • Hybridization conditions that allow this differential hybridization can be devised by methods known in the art. Futhermore, specific sequences that hybridize to GFAT II and not to GFAT can be deduced from the sequences provided using algorithms available in the art.
  • the consensus sequences identified in Fig. 3 and Fig. 4 can also be used to generate nucleic acids of the invention.
  • the consensus sequence of Fig. 3 or one encoding the consensus sequence of Fig. 4, or a fragment of either can be used to generate hybridization or PCR probes and they can also be used to search databases of expressed and genomic sequences.
  • a nucleic acid comprising the consensus sequence of Fig 3 or encoding the consensus sequence of Fig. 4 can also be produced and used in other ways, such as in producing a protein, peptide, fusion protein or variant of the invention.
  • the present invention also provides a substantially pure GFAT II protein, or fragment thereof, comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, 6, or 8; an amino acid sequence encoded by a nucleic acid that specifically hybridizes to the complement of SEQ ID NO: 3, 5, 7, especially at high stringency conditions; a fragment of SEQ ID NO: 4, 6, or 8, containing at least about 15 to about 36 contiguous amino acids that are not identical to a known GFAT sequence, or about 50 to about 100 contiguous amino acids of SEQ ID NO: 4, 6, or 8.
  • variant GFAT II proteins or fragments can be generated by known mutagenesis techniques so that one or more amino acids can be substituted, deleted, or added and the GFAT activity retained.
  • the present invention also provides an antibody capable of specifically binding to a substantially pure GFAT II protein, fragment, or variant.
  • the antibody can be monoclonal or polyclonal.
  • the invention also provides numerous possible methods for producing GFAT II proteins, fragments, fusion proteins, and peptides. These methods can encompass recombinant DNA techniques, homologous recombination techniques, transgenic animal techniques,or gene activation techniques. Cells generated as a result of these techniques are also provided, as well as progeny of these cells and organisms possessing these cells.
  • the present invention provides a transformed cell possesing a nucleic acid molecule of the invention, such as one comprising SEQ ID NO: 3, 5, or 7, or a fragment thereof, one comprising a nucleic acid enoding a GFAT II protein or fragment, such as in SEQ ID NO: 4, 6, or 8, or a nucleic acid that hybridizes to one or more of SEQ ID NO: 3, 5, or 7, or complements thereof, especially under stringent conditions.
  • the nucleic acid can be introduced into the cell via a recombinant vector and a tranformation process, by homologous recombination, by microinjection, liposome fusion, or other known means.
  • the cell can be any appropriate host.
  • Examples include a mammalian cell, either an established cell line or a primary cultured cell, a bacteria cell, an insect cell, a yeast cell, or a cell within an organism, such as a hepatic or skeletal muscle cell of a mammal.
  • the proteins, fragments, fusion proteins, and cells of the invention can be used in methods to screen for compounds or compositions that effect GFAT II enzyme activity.
  • a specific inhibitor of GFAT II can be identified using an assay comprising a GFAT II protein, fragment, fusion protein, or a cell containing a GFAT II protein, fragment, or fusion protein, adding a test compound or composition, and comparing GFAT activity to a control.
  • a specific GFAT II inhibitors can be identified using these methods.
  • compounds or compositions the specifically or preferentially effect GFAT can be identified.
  • assays for compounds and compositions that promote, reduce, irreversibly inhibit, or reversibly inhibit GFAT activity in a GFAT II protein, fragment or fusion protein can be screened for.
  • the invention provides a transformed cell having a nucleic acid molecule which comprises: (A) an exogenous promoter region which functions in a cell to cause the production of an mRNA molecule; which is linked to (B) a structural nucleic acid molecule, wherein the structural nucleic acid molecule encodes a GFAT II gene or fragment thereof, which is linked to (C) a 3' non-translated sequence that functions in a cell to cause termination of transcription and addition of polyadenylated ribonucleotides to a 3' end of the mRNA molecule.
  • the invention also provides numerous methods for assaying or determining the level or pattern of a GFAT II protein or mRNA expressed or present in a cell, organism, tissue, or biological fluid.
  • the invention provides a method for determining a level or pattern of a GFAT II protein or mRNA expression in a cell comprising: (A) incubating, under conditions permitting nucleic acid hybridization, a marker nucleic acid molecule, the marker nucleic molecule capable of specifically hybridizing to a nucleic acid molecule that encodes GFAT II or complement thereof under high stringency conditions and the marker nucleic acid molecule incapable of specifically hybridizing to a nucleic acid molecule that encodes GFAT or complement thereof under high stringency conditions, with a complementary nucleic acid molecule obtained from the cell, wherein nucleic acid hybridization between the marker nucleic acid molecule, and the complementary nucleic acid molecule obtained from the cell permits the detection of GFAT II; (B) permitting hybridization between the marker nucleic acid molecule and
  • the present invention also provides a method for determining a mutation in a cell whose presence is predictive of a mutation affecting the level or pattern of a GFAT II protein comprising the steps: (A) incubating, under conditions permitting nucleic acid hybridization, a marker nucleic acid molecule, the marker nucleic acid molecule comprising a nucleic acid molecule that is linked to a gene, the gene specifically hybridizes to a nucleic acid molecule having a nucleic acid sequence having SEQ ID NO: 4 or the complement thereof, and a complementary nucleic acid molecule obtained from the cell, wherein nucleic acid hybridization between the marker nucleic acid molecule and the complementary nucleic acid molecule obtained from the cell permits the detection of a polymorphism whose presence is predictive of a mutation affecting the level or pattern of the GFAT II protein in the cell; (B) permitting hybridization between the marker nucleic acid molecule and the complementary nucleic acid molecule obtained from the cell; and (C) detecting the presence of the
  • the present invention also provides a process for diagnosis or prognosis of insulin resistance in a non-insulin dependent diabetes mellitus mammal, which comprises assaying the concentration of a molecule, whose concentration is dependent upon the expression of a GFAT II gene, the molecule being present in or derived from a sample of cells or bodily fluid of the mammal, comparing the concentration of that molecule present in a sample of cells or bodily fluid of a mammal that does not have insulin resistant non-insulin dependent diabetes mellitus and is not predisposed to developing insulin resistance, wherein a concentration of the molecule differs from that found in the mammal who does not have insulin resistance and is not predisposed to having insulin resistance is diagnostic or prognostic of insulin resistance in the patient.
  • the present invention also provides a method for diagnosing glucose intolerance in a patient which comprises the steps: (A) incubating under conditions permitting nucleic acid hybridization: a marker nucleic acid molecule, the marker nucleic acid molecule comprising a nucleotide sequence of a polynucleotide that specifically hybridizes to a polynucleotide that is linked to a GFAT II gene, and a complementary nucleic acid molecule obtained from a cell or a bodily fluid of the patient, wherein nucleic acid hybridization between the marker nucleic acid molecule, and the complementary nucleic acid molecule obtained from the patient permits the detection of a polymorphism whose presence is predictive of a mutation affecting GFAT II response in the patient; (B) permitting hybridization between the marker nucleic acid molecule and the complementary nucleic acid molecule obtained from the patient; and (C) detecting the presence of the polymorphism, wherein the detection of the polymorphism is diagnostic of insulin resistance.
  • the present invention also provides a method of determining an association between a polymorphism and a trait comprising: (A) hybridizing a nucleic acid molecule specific for the polymorphism to genetic material of a cell, wherein the nucleic acid molecule has a nucleic acid sequence of SEQ ID NO: 4 or complements thereof; and (B) calculating the degree of association between the polymorphism and the trait.
  • the present invention also provides a method for diagnosing insulin resistant NIDDM in a patient which comprises the steps: (A) incubating under conditions permitting nucleic acid hybridization: a marker nucleic acid molecule, the marker nucleic acid molecule comprising a nucleotide sequence of a polynucleotide that specifically hybridizes to a polynucleotide that is linked to a sequence that specifically hybridizes to a gene that encodes the protein of GFAT II gene, and a complementary nucleic acid molecule obtained from a cell or a bodily fluid of the patient, wherein nucleic acid hybridization between the marker nucleic acid molecule, and the complementary nucleic acid molecule obtained from the patient permits the detection of a polymorphism whose presence is predictive of a mutation affecting the level or pattern of the protein of GFAT II in the patient; (B) permitting hybridization between the marker nucleic acid molecule and the complementary nucleic acid molecule obtained from the patient; and (C) detecting the presence of the
  • the present invention also provides a method of producing a cell containing an overexpressed GFAT II protein comprising: (A) transforming the cell with a functional nucleic acid molecule, wherein the functional nucleic acid molecule comprises a promoter region, wherein the promoter region is linked to a structural region, wherein the structural region comprises a nucleic acid molecule having a nucleic acid sequence of SEQ ID NO: 3, wherein the structural region is linked to a 3' non-translated sequence that functions in the cell to cause termination of transcription and addition of polyadenylated ribonucleotides to a 3' end of a mRNA molecule; and wherein the functional nucleic acid molecule results in overexpression of the GFAT II protein; and (B) culturing the transformed cell.
  • the invention also provides a cell and progeny of a cell produced by such a method.
  • the present invention also provides a method of producing a cell containing reduced levels of a GFAT II protein comprising: (A) transforming the cell with a functional nucleic acid molecule, wherein the functional nucleic acid molecule comprises a promoter region, wherein the promoter region is linked to a structural region, wherein the structural region comprises a nucleic acid molecule having a nucleic acid sequence of SEQ ID NO: 4, wherein the structural region is linked to a 3' non-translated sequence that functions in the cell to cause termination of transcription and addition of polyadenylated ribonucleotides to a 3' end of a mRNA molecule; and wherein the functional nucleic acid molecule results in co-suppression of the GFAT protein; and (B) culturing the transformed cell.
  • the invention also provides a cell and progeny of a cell produced by such a method.
  • the present invention also provides a method for reducing expression of a GFAT II protein in a cell comprising: (A) transforming the cell with a nucleic acid molecule, the nucleic acid molecule having an exogenous promoter region which functions in a cell to cause the production of a mRNA molecule, wherein the exogenous promoter region is linked to a transcribed nucleic acid molecule having a transcribed strand and a non-transcribed strand, wherein the transcribed strand is complementary to a nucleic acid molecule having a nucleic acid sequence of SEQ ID NO: 4 or its complement and the transcribed strand is complementary to an endogenous mRNA molecule; and wherein the transcribed nucleic acid molecule is linked to a 3' non-translated sequence that functions in the cell to cause termination of transcription and addition of polyadenylated ribonucleotides to a 3' end of a mRNA molecule; and (B) culturing the transformed cell
  • the present invention also provides a method of isolating a nucleic acid that encodes a GFAT II protein or fragment thereof comprising: (A) incubating under conditions permitting nucleic acid hybridization, a first nucleic acid molecule comprising SEQ ID NO: 4 or the complement thereof with a complementary second nucleic acid molecule obtained from a cell; (B) permitting hybridization between the first nucleic acid molecule and the second nucleic acid molecule obtained from the cell; and (C) isolating the second nucleic acid molecule.
  • the invention also provides a cell and progeny of a cell produced by such a method.
  • the present invention also provides a method of producing a cell containing an overexpressed GFAT II gene comprising: (A) transforming the cell with a functional nucleic acid molecule, wherein the functional nucleic acid molecule comprises a promoter region, wherein the promoter region is linked to a structural region, wherein the structural region comprises a nucleic acid molecule having a nucleic acid sequence consisting of SEQ ID NO:4 or complements thereof; wherein the structural region is linked to a 3' non-translated sequence that functions in the cell to cause termination of transcription and addition of polyadenylated ribonucleotides to a 3' end of a mRNA molecule; and wherein the functional nucleic acid molecule results in overexpression of the GFAT II gene protein; and (B) culturing the transformed cell. .
  • the invention also provides a cell and progeny of a cell produced by such a method.
  • the invention also provides a cell and progeny of a cell produced by such
  • the present invention also provides a method of producing a cell containing reduced levels of a GFAT II gene comprising: (A) transforming the cell with a functional nucleic acid molecule, wherein the functional nucleic acid molecule comprises a promoter region, wherein the promoter region is linked to a structural region, wherein the structural region comprises a nucleic acid molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 4 or complements thereof; wherein the structural region is linked to a 3' non-translated sequence that functions in the cell to cause termination of transcription and addition of polyadenylated ribonucleotides to a 3' end of a mRNA molecule; and wherein the functional nucleic acid molecule results in co-suppression of the GFAT II gene; and (B) culturing the transformed cell.
  • the invention also provides a cell and progeny of a cell produced by such a method.
  • the present invention also provides a method for reducing expression of a GFAT II gene in a cell comprising: (A) transforming the cell with a nucleic acid molecule, the nucleic acid molecule having an exogenous promoter region which functions in a cell to cause the production of a mRNA molecule, wherein the exogenous promoter region is linked to a transcribed nucleic acid molecule having a transcribed strand and a non-transcribed strand, wherein the transcribed strand is complementary to a nucleic acid molecule having a nucleic acid selected from the group consisting of SEQ ID NO:4 or complements thereof and the transcribed strand is complementary to an endogenous mRNA molecule; and wherein the transcribed nucleic acid molecule is linked to a 3' non-translated sequence that functions in the cell to cause termination of transcription and addition of polyadenylated ribonucleotides to a 3' end of a mRNA molecule; and (B)
  • the present invention also provides a method of using a GFAT II protein or fragment thereof in an assay for screening test substances for the ability to modulate or maintain GFAT II activity.
  • Fig. 1 diagramatically sets forth a comparison between the nucleic acid sequences that encode GFAT and the nucleic acid sequence that encode GFAT II.
  • Fig. 2 diagramatic lly sets forth a comparison between the amino acid sequences of GFAT and GFAT II.
  • Fig. 3 diagramatically sets forth a comparison between the nucleic acid sequences that encode human, murine, and rat GFAT II, and the consensus sequence of the three.
  • Fig. 4 diagramatically sets forth a comparison between the amino acid sequences of human, murine, and rat GFAT II, and the consensus sequence of the three.
  • altered means that expression differs from the expression response of cells or tissues not exhibiting the phenotype.
  • amino acid(s) means all naturally occurring L- amino acids.
  • biologically active means activity with respect to either a structural or a catalytic attribute, which includes the capacity of a nucleic acid to hybridize to another nucleic acid molecule, or the ability of a protein to be bound by an antibody (or to compete with another molecule for such binding), among others.
  • Catalytic attributes involve the capacity of the agent to mediate a chemical reaction or response.
  • chromosome walking means a process of extending a genetic map by successive hybridization steps.
  • cluster means that BLAST scores from pairwise sequence comparisons of the member clones are similar enough to be considered identical with experimental error.
  • complement means that one nucleic acid exhibits complete complementarity with another nucleic acid.
  • complementarity means that two molecules can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional high stingency conditions.
  • degenerate means that two nucleic acid molecules encode for the same amino acid sequences but comprise different nucleotide sequences.
  • exogenous genetic material means any genetic material, whether naturally occurring or otherwise, from any source that is capable of being inserted into any organism.
  • expansion means the differentiation and proliferation of cells.
  • expression response means the mutation affecting the level or pattern of the expression encoded in part or whole by one or more nucleic acid molecules.
  • fragment means a nucleic acid molecule whose sequence is shorter than the target or identified nucleic acid molecule and having the identical, the substantial complement, or the substantial homologue of at least 10 contiguous nucleotides of the target or identified nucleic acid molecule.
  • fusion molecule means a protein-encoding molecule or fragment that upon expression, produces a fusion protein.
  • fusion protein means a protein or fragment thereof that comprises one or more additional peptide regions not derived from that protein.
  • GFAT activitity can be determined or detected in a number of ways. For example, conversion of fructose-6-phosphate to glucosamine-6-phosphate can be followed by separating the two compounds on an anion exchange resin.
  • Buse et al. (Amer, J. Physiol. 272: E1080-88 (1997)) describes a method of fiuorometrically measuring glucosamine-6-phosphate after derivitvation with o- phthaladehyde and separation by HPLC that can be used.
  • Marshall et al. J. Biol. Chem.
  • hybridization probe means any nucleic acid capable of being labeled and forming a double-stranded structure with another nucleic acid over a region large enough for the double stranded structure to be detected.
  • isolated means an agent is separated from another specific component with which it occurred.
  • the isolate material may be purified to essential homogeneity, as determined by PAGE or column chromatography, such as HPLC.
  • An isolated nucleic acid can comprise at least about 50, 80, or 90% (on a molar basis) of all macromolecular species present.
  • marker nucleic acid means a nucleic acid molecule that is utilized to determine an attribute or feature (e.g., presence or absence, location, correlation, etc.) of a molecule, cell, or tissue.
  • trimer refers to a compound having similar functional and/or structural properties to another known compound or a particular fragment of that known compound.
  • minimum complementarity means that two molecules can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional low stringency conditions.
  • PCR probe means a nucleic acid capable of initiating a polymerase activity while in a double-stranded structure with another nucleic acid.
  • Krzesicki, et al Am. J. Respir. Cell Mol Biol. 16:693-701 (1997), incorporated by reference in its entirety, discusses the preparation of PCR probes for use in identifying nucleic acids of osteoarthrits tissue. Other methods for determining the structure of PCR probes and PCR techniques have been described.
  • phenotype means any of one or more characteristics of an organism, tissue, or cell.
  • primer means a single-stranded oligonucleotide which acts as a point of initiation of template-directed DNA synthesis under appropriate conditions (e.g., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as, DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.
  • the appropriate length of a primer depends on the intended use of the primer, but typically ranges from 15 to 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.
  • a primer need not reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with a template.
  • probe means an agent that is utilized to determine an attribute or feature (e.g. presence or absence, location, correlation, etc.) of a molecule, cell, tissue, or organism.
  • protein fragment means a peptide or polypeptide molecule whose amino acid sequence comprises a subset of the amino acid sequence of that protein.
  • protein molecule/peptide molecule means any molecule that comprises five or more amino acids.
  • recombinant means any agent (e.g., DNA, peptide, etc.), that is, or results from, however indirectly, human manipulation of a nucleic acid molecule.
  • the recombination may occur inside a cell or in a tube.
  • selectable or screenable marker genes means genes who's expression can be detected by a probe as a means of identifying or selecting for transformed cells.
  • sensingleton means a single clone.
  • the term “specifically bind” means that the binding of an antibody or peptide is not competitively inhibited by the presence of non-related molecules.
  • the term “specifically hybridizing” means that two nucleic acid molecules are capable of forming an anti-parallel, double-stranded nucleic acid structure.
  • substantially complement means that a nucleic acid sequence shares at least 80% sequence identity with the complement.
  • substantially fragment means a fragment which comprises at least 100 nucleotides.
  • substantially homologue means that a nucleic acid molecule shares at least 80% sequence identity with another.
  • substantially identical means that 70% to about 99% of a region or fragment in a molecule is identical to a region of a different molecule.
  • individual units e.g., nucleotides or amino acids
  • a percentage identity of the units identical over the total number of units in the region is determined.
  • Numerous algorithmic and computerized means for determining a percentage identity are known in the art. These means may allow for gaps in the region being considered in order to produce the highest percentage identity.
  • substantially hybridizing means that two nucleic acid molecules can form an anti-parallel, double-stranded nucleic acid structure under conditions (e.g. salt and temperature) that permit hybridization of sequences that exhibit 90% sequence identity or greater with each other and exhibit this identity for at least a contiguous 50 nucleotides of the nucleic acid molecules.
  • tissue sample means any sample that comprises more than one cell.
  • the present invention stems in part from the isolation of a cDNA molecule that encodes human GFAT II, which exhibits nucleic acid and amino acid sequence similarity to GFAT. As the new protein exhibits a similar primary sequence to GFAT, it is referred to herein as "GFAT II.”
  • SEQ ID NO: 4 sets forth the amino acid sequence of human GFAT II
  • SEQ ID NO: 3 sets forth the nucleic acid sequence of human GFAT II.
  • Figure 1 diagramatically sets forth a comparison between the nucleic acid sequences of GFAT and GFAT II.
  • Figure 2 diagramatically sets forth a comparison between the amino acid sequences encoding GFAT and GFAT II.
  • In situ hybridization shows that the human GFAT II gene is located on chromosome 5q33.5-37.5, preferably 5q 34.5-36.5, and even more preferably at 5q35.5.
  • the human GFAT gene is located on chromosome 2pl3 (Zhou et al, Human Genet. 96:99-101 (1995)).
  • GFAT II and GFAT exhibit different expression profiles when varies tissues were sampled.
  • the expression patterns of GFAT and GFAT II are set forth in Example 2.
  • NIDDM has its art-recognized meaning.
  • the methods of the present invention are particularly relevant in the monitoring of the expression of GFAT II.
  • the methods of the present invention are also relevant in the monitoring of the expression of GFAT II in NIDDM patients.
  • Molecules of the present invention are capable of being used to diagnose GFAT II expression.
  • Molecules of the present invention are also capable of being used to diagnose the level of GFAT II expression in NIDDM patients.
  • Molecules of the present invention can also be used as therapeutic agents and in diagnostic methods.
  • the molecules of the present invention may be either naturally occurring or non-naturally occurring.
  • a naturally occurring molecule may be "substantially pure” or “substantially purified,” if desired, such that one or more molecules that is or may be present in a naturally occurring preparation containing that molecule will have been removed or will be present at a lower concentration than that at which it would normally be found.
  • the molecules of the present invention comprise nucleic acid molecules, proteins, antibodies, and organic molecules.
  • A. Nucleic Acid Molecules A preferred class of agents of the present invention comprise GFAT II nucleic acid molecules. Such molecules may be, for example, DNA or RNA.
  • such nucleic acid molecules will encode all or a fragment of GFAT II protein, preferably a human, mouse, or rat GFAT II protein, as well as, optionally contain its "promoter” or flanking gene sequences.
  • promoter is used in an expansive sense to refer to the regulatory sequence(s) that control mRNA production. Such sequences include RNA polymerase binding sites, enhancers, etc. All such GFAT II molecules may be used in a diagnostic or therapeutic context.
  • Fragment GFAT II nucleic acid molecules may encode significant portion(s) of, or indeed most of, the GFAT II protein.
  • a fragment GFAT II nucleic acid molecule is identical or complementary to at least about 20 contiguous nucleotides in SEQ ID NO: 3. More preferably it comprises at least about 25 nucleotides in SEQ ID NO: 3. Even more preferably it comprises at least about 50 nucleotides in SEQ ID NO: 3. Most preferably, it comprises at least about 100 nucleotides in SEQ ID NO: 3.
  • a fragment GFAT II nucleic acid molecule comprises at least one nucleotide that is not found in a corresponding position in GFAT II.
  • the fragment GFAT II protein exhibits GFAT II enzymatic activity.
  • GFAT II nucleic acid molecules and fragment GFAT II nucleic acid molecules can specifically hybridize with other nucleic acid molecules.
  • two nucleic acid molecules are said to be capable of specifically hybridizing to one another if the two molecules are capable of forming an anti-parallel, double-stranded nucleic acid structure.
  • Appropriate stringency conditions which promote DNA hybridization are, for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1- 6.3.6.
  • the salt concentration in the wash step can be selected from a moderately low stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2 x SSC at 50°C.
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C. Both temperature and salt may be varied, or either the temperature or the salt concentration may be held constant while the other variable is changed.
  • a nucleic acid of the present invention will specifically hybridize to a nucleic acid molecule having SEQ ID NO: 3 or its complement under moderately stringent conditions, for example at about 2.0 x SSC and about 40°C about 50°C.
  • a nucleic acid of the present invention will specifically hybridize to SEQ ID NO: 3 or its complement under high stringency conditions, such as about 0.2 x 55C and about 45°C to about °65C.
  • a nucleic acid molecule of the present invention will comprise SEQ ID NO: 3 or its complement.
  • Fragment nucleic acid molecules can be determined and selected such that, under specified conditions, such as high stringency, they can be used to specifically hybridize to GFAT II sequences and not, for example, to GFAT sequences. Algorithms for determining these fragments nucleic acid molecules are known and available.
  • a nucleic acid molecule of the present invention shares between about 100% and about 70% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 3 or its complement. In a further aspect of the present invention, a nucleic acid molecule of the present invention shares between about 100% and about 95% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 3 or its complement. In another aspect of the present invention, a nucleic acid molecule of the present invention shares between about 100% and about 98% sequence identity with SEQ ID NO: 3 or its complement.
  • the degeneracy of the genetic code which allows different nucleic acid sequences to code for the same protein or peptide, is known in the literature. (U.S. Patent No.
  • nucleic acid molecule is degenerate of another nucleic acid molecule when the nucleic acid molecules encode for the same amino acid sequences, but comprise different nucleotide sequences.
  • nucleic acid molecules of the present invention include nucleic acid molecules that are degenerate of SEQ ID NO: 3, 5, or 7 and its complement.
  • the nucleic acid molecules of the present invention can be employed to obtain other GFAT II molecules. Nucleic acid molecules and fragments thereof of the present invention may be employed to obtain other nucleic acid molecules from humans.
  • nucleic acid molecules include the nucleic acid molecules that encode the complete coding sequence of a protein and promoters and flanking sequences of such molecules.
  • nucleic acid molecules include nucleic acid molecules that encode for other isozymes or gene family members.
  • Such molecules can be readily obtained by using the above-described nucleic acid molecules, or fragments thereof, to screen cDNA or genomic libraries obtained from humans or to search databases of sequence information. Methods for forming such libraries and searching databases are well known in the art.
  • Nucleic acid molecules and fragments thereof of the present invention may also be employed to obtain nucleic acid homologues.
  • Such homologues include the nucleic acid molecule of other organisms (e.g., monkey, dog, cat) including the nucleic acid molecules that encode, in whole or in part, protein homologues of other species or other organisms, and sequences of genetic elements such as promoters and transcriptional regulatory elements from other species or organisms.
  • Such molecules can be readily obtained by using the above-described nucleic acid molecules or fragments thereof to screen cDNA or genomic libraries obtained from such species. Methods for forming such libraries are well known in the art.
  • Such homologue molecules may differ in their nucleotide sequences from those found SEQ ID NO: 3 or complement thereof.
  • the nucleic acid molecules of the present invention therefore also include molecules that, although capable of specifically hybridizing with the nucleic acid molecules may lack "complete complementarity.”
  • the nucleic acid molecules of the present invention include molecules that encode the murine GFAT II protein, its promoter, or flanking gene sequences.
  • a murine nucleic acid of the present invention will specifically hybridize to a nucleic acid molecule having SEQ ID NO: 5 or its compliment.
  • a murine nucleic acid molecule of the present invention will comprise SEQ ID NO: 5 or its compliment.
  • Fragment murine GFAT II nucleic acid molecules may encode significant portion(s) of, or indeed most of, the murine GFAT II protein.
  • a fragment GFAT II nucleic acid molecule may be identical or complementary to at least about 20 contiguous nucleotides in SEQ ID NO: 5. In another embodiment, it comprises at least aboutt 25 nucleotides in SEQ ID NO: 5. In a further embodiment, it comprises at least about 50 nucleotides in SEQ ID NO: 5. In yet another embodiment, it comprises at least about 100 nucleotides in SEQ ID NO: 5.
  • a fragment murine GFAT II nucleic acid molecule may comprises at least one nucleotide that is not found in a corresponding position in murine GFAT II.
  • the fragment murine GFAT II molecule exhibits murine GFAT II enzymatic activity.
  • a nucleic acid molecule of the present invention shares between about 100% and about 70% sequence identity with the murine nucleic acid sequence set forth in SEQ ID NO: 5 or its complement. In a further aspect of the present invention, a nucleic acid molecule of the present invention shares between about 100% and about 95% sequence identity with the murine nucleic acid sequence set forth in SEQ ID NO: 5 or its complement. In another aspect of the present invention, a nucleic acid molecule of the present invention shares between about 100% and about 98% sequence identity with SEQ ID NO: 5 or its complement.
  • the present invention also provides a substantially pure murine GFAT II protein or fragment thereof encoded by a nucleic acid sequence which specifically hybridizes to SEQ ID NO: 6 or its complement. The present invention also provides a substantially pure murine GFAT II protein having SEQ ID NO: 6.
  • the present invention also provides a substantially pure murine GFAT II protein or fragment thereof comprising at least 37 consecutive amino acids of SEQ ID NO: 6.
  • the present invention also provides an antibody capable of specifically binding to the substantially pure murine GFAT II protein.
  • the nucleic acid molecules of the present invention include molecules that encode the rat GFAT II protein, its promoter, or flanking gene sequences.
  • a rat nucleic acid of the present invention will specifically hybridize to a nucleic acid molecule having SEQ ID NO: 7 or its compliments.
  • a rat nucleic acid molecule of the present invention will comprise SEQ ID NO: 7 or its compliment.
  • Fragment rat GFAT II nucleic acid molecules may encode significant portion(s) of, or indeed most of, the rat GFAT II protein.
  • a fragment rat GFAT II nucleic acid molecule may be identical or complementary to at least about 20 contiguous nucleotides in SEQ ID NO: 7. In another aspect, it comprises at least about 25 nucleotides in SEQ ID NO: 7. In a further aspect, it comprises at least about 50 nucleotides in SEQ ID NO: 7. In yet another aspect, it comprises at least about 100 nucleotides in SEQ ID NO: 7.
  • a fragment rat GFAT II nucleic acid molecule comprises at least one nucleotide that is not found in a corresponding position in rat GFAT II. In yet another embodiment, the fragment rat GFAT II molecule exhibits rat GFAT II enzymatic activity.
  • a nucleic acid molecule of the present invention shares between about 100% and about 70% sequence identity with the rat nucleic acid sequence set forth in SEQ ID NO: 7 or its complement. In a further aspect of the present invention, a nucleic acid molecule of the present invention shares between about 100% and about 95% sequence identity with the rat nucleic acid sequence set forth in SEQ ID NO: 7 or its complement. In another aspect of the present invention, a nucleic acid molecule of the present invention shares between about 100% and about 98% sequence identity with SEQ ID NO: 7 or its complement.
  • the present invention also provides a substantially pure rat GFAT II protein or fragment thereof encoded by a nucleic acid sequence which specifically hybridizes to SEQ ID NO: 8 or its complement.
  • the present invention also provides a substantially pure rat GFAT II protein having SEQ ID NO: 8.
  • the present invention also provides a substantially pure rat GFAT II protein or fragment thereof comprising at least 37 consecutive amino acids of SEQ ID NO: 8.
  • the present invention also provides an antibody capable of specifically binding to the substantially pure rat GFAT II protein.
  • Nucleic acid molecules of the present invention may be used to find any rodent
  • GFAT II nucleic acid homologue Nucleic acid molecules of the present invention may also be used to obtain any mammalian GFAT II homologue.
  • the GFAT II promoter sequence(s) and GFAT II flanking sequences can also be obtained using the SEQ ID NO: 3, 5, or 7 sequence provided herein. In one embodiment, such sequences are obtained by incubating oligonucleotide probes of GFAT II oligonucleotides with members of genomic human libraries and recovering clones that hybridize to the probes. In a second embodiment, methods of
  • chromosome walking or 3' or 5' RACE may be used (Frohman, M.A. et al, Proc. Natl. Acad. Sci. (U.S.A.) 55:8998-9002 (1988); Ohara, O. et al, Proc. Natl Acad. Sci. (U.S.A.) 56:5673-5677 (1989)) to obtain such sequences.
  • B. Proteins and Peptides A second class of preferred agents comprises GFAT II protein, its peptide fragments, fusion proteins, and analogs. GFAT II protein may be produced via chemical synthesis, or more alternatively, by expressing GFAT Il-encoding cDNA in a suitable bacterial or eukaryotic host.
  • GFAT II subsequence of such cDNA that encodes GFAT II may be used for this purpose (SEQ ID NO: 3). Suitable methods for expression are described by Sambrook, J., et al, (In: Molecular Cloning, a Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, New York (1989)), or similar texts.
  • a "GFAT II fragment” is a peptide or polypeptide whose amino acid sequence comprises a subset of the amino acid sequence of GFAT II protein.
  • a fragment GFAT II molecule is identical or complementary to at least one region which corresponds to a contiguous 37 amino acids of SEQ ID NO: 4; more preferably, at least one region which corresponds to a contiguous 50 amino acids of SEQ ID NO: 4; in another embodiment, at least one region which responds to a contiguous 100 amino acids of SEQ ID NO: 4.
  • a GFAT II protein or fragment thereof that comprises one or more additional non-GFAT II peptide regions or amino acids is a "GFAT II fusion" protein.
  • Such molecules may be derivatized to contain carbohydrate or other moieties (such as keyhole limpet hemocyanin, etc.).
  • the fragments and fusions of the present invention are preferably produced via recombinant means.
  • the analogs of the GFAT II molecules comprise GFAT II proteins, fragments or fusions in which non-essential, or non-relevant, amino acid residues have been added, replaced, or deleted.
  • An example of such an analog is the GFAT II protein of non-human species, such as primates, dogs, cats, etc.
  • Such analogs can readily be obtained by any of a variety of methods.
  • the disclosed SEQ ID NO:3 will be used to define a pair of primers that may be used to isolate the GFAT Il-encoding nucleic acid molecules from any desired species. Such molecules can be expressed to yield GFAT II analogs by recombinant means.
  • One aspect of the present invention concerns antibodies, single-chain antigen binding molecules, or other proteins that specifically bind to GFAT II protein and its analogs, fusions or fragments.
  • Such antibodies are "anti-GFAT II antibodies,” and may be used, for example to measure GFAT II protein.
  • an antibody or peptide is said to "specifically bind" to GFAT II if such binding is not competitively inhibited by the presence of non-GFAT II molecules.
  • Nucleic acid molecules that encode all or part of the GFAT II protein can be expressed, via recombinant means, to yield GFAT II protein or peptides that can in turn be used to elicit antibodies that are capable of binding GFAT II.
  • Such antibodies may be used in immunodiagnostic assays.
  • Such GFAT Il-encoding molecules, or their fragments may be a "fusion" molecule (i.e. a part of a larger nucleic acid molecule) such that, upon expression, a fusion protein is produced.
  • the antibodies that specifically bind GFAT II proteins and protein fragments may be polyclonal or monoclonal, and may comprise intact immunoglobulins, of antigen binding portions of immunoglobulins (such as (F(ab'), F(ab')2) fragments, or single-chain immunoglobulins producable, for example, via recombinant means. It is understood that practitioners are familiar with the standard resource materials which describe specific conditions and procedures for the construction, manipulation and isolation of antibodies (see, for example, Harlow and Lane, In Antibodies: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York (1988), the entirety of which is herein incorporated by reference).
  • Murine monoclonal antibodies are particularly preferred. BALB/c mice are preferred for this purpose, however, equivalent strains may also be used.
  • the animals are immunized with, for example, approximately 25 ⁇ g of purified GFAT II protein (or fragment thereof) that has been emulsified with a suitable adjuvant (such as TiterMax adjuvant (Vaxcel, Norcross, GA)).
  • a suitable adjuvant such as TiterMax adjuvant (Vaxcel, Norcross, GA)
  • Immunization may be conducted at two intramuscular sites, one intraperitoneal site, and one subcutaneous site at the base of the tail.
  • An additional i.v. injection of approximately 25 ⁇ g of antigen is preferably given in normal saline three weeks later. After approximately 11 days following the second injection, the mice may be bled and the blood screened for the presence of anti-GFAT II antibodies.
  • a direct binding ELISA is employed for this purpose.
  • the mouse having the highest antibody titer is given a third i.v. injection of approximately 25 ⁇ g of GFAT II protein or fragment.
  • the splenic leukocytes from this animal may be recovered 3 days later, and are then permitted to fuse, for example, using polyethylene glycol, with cells of a suitable myeloma cell line (such as, for example, the P3X63Ag8.653 myeloma cell line).
  • Hybridoma cells are selected by culturing the cells under "HAT" (hypoxanthine- aminopterin-thymine) selection for about one week.
  • the resulting clones may then be screened for their capacity to produce monoclonal antibodies ("mAbs) to GFAT II protein, preferably by direct ELISA.
  • anti-GFAT II monoclonal antibodies are isolated using GFAT II fusions, or conjugates, as immunogens.
  • a group of mice can be immunized using a GFAT II fusion protein emulsified in Freund's complete adjuvant (approximately 50 ⁇ g of antigen per immunization).
  • an identical amount of antigen is emulsified in Freund's incomplete adjuvant and used to immunize the animals.
  • serum samples are taken and evaluated for the presence of antibody. If antibody titers are too low, a fourth booster can be employed. Polysera capable of binding GFAT II at 1 :5,000 dilution can also be obtained using this method.
  • the spleens of the above-described immunized mice are removed, disrupted, and immune splenocytes are isolated over a ficoll gradient.
  • the isolated splenocytes are fused, using polyethylene glycol with BALB/c-derived HGPRT (hypoxanthine guanine phosphoribosyl transferase) deficient P3x63xAg8.653 plasmacytoma cells.
  • the fused cells are plated into 96 well microtiter plates and screened for hybridoma fusion cells by their capacity to grow in culture medium supplemented with hypothanthine, aminopterin and thymidine for approximately 2-3 weeks.
  • out of every 10° " spleen cells subjected to fusion yields a viable hybridoma.
  • a typical spleen yields 5-10 x l ⁇ 7 spleen cells.
  • Hybridoma cells that arise from such incubation are preferably screened for their capacity to produce an immunoglobulin that binds to GFAT II protein.
  • An indirect ELISA may be used for this purpose.
  • the supernatants of hybridomas are incubated in microtiter wells that contain immobilized GFAT II protein. After washing, the titer of bound immunoglobulin can be determined using, for example, a goat anti-mouse antibody conjugated to horseradish peroxidase. After additional washing, the amount of immobilized enzyme is determined (for example, through the use of a chromogenic substrate).
  • Such screening is performed as quickly as possible after the identification of the hybridoma in order to ensure that a desired clone is not overgrown by non-secreting neighbors.
  • the fusion plates are screened several times since the rates of hybridoma growth vary.
  • a different antigenic form of GFAT II may be used to screen the hybridoma.
  • the splenocytes may be immunized with one GFAT II immunogen, but the resulting hybridomas can be screened using a different GFAT II immunogen.
  • antibody molecules or their fragments may be used for diagnostic purposes.
  • a ligand group such as biotin
  • a detectable marker group such as fluorescent group, a radioisotope or an enzyme
  • a "mimetic compound" of GFAT II is, under on definition, a compound that is not GFAT II, or a fragment of GFAT II, but which nonetheless exhibits an ability to specifically bind to anti-GFAT II antibodies. Such molecules can be used to elicit anti-GFAT II antibodies, and thus, may be used to assist diagnosis of GFAT II related disorders. III. Uses of the Molecules of the Invention
  • An aspect of the present invention provides plasmid DNA vectors for use in the expression of the GFAT II protein.
  • These vectors contain the DNA sequences described above which code for the polypeptides of the invention.
  • Appropriate vectors which can transform eukaroytic cells, including mammalian cells and microorganisms capable of expressing the GFAT II protein include expression vectors comprising nucleotide sequences coding for the GFAT II protein joined to transcriptional and translational regulatory sequences which are selected according to the host cells used.
  • Vectors incorporating modified sequences as described above are included in the present invention and are useful in the production of the GFAT II polypeptides.
  • the vector employed in the method also contains selected regulatory sequences in operative association with the DNA coding sequences of the invention which are capable of directing the replication and expression thereof in selected host cells. Transfer of a nucleic acid that encodes for a protein can result in overexpression of that protein in a transformed cell.
  • One or more of the proteins or fragments thereof encoded by nucleic acid molecules of the present invention may be overexpressed in a transformed cell.
  • any of the GFAT II proteins or fragments thereof may be overexpressed in a transformed cell.
  • Such overexpression may be the result of transient or stable transfer of the exogenous genetic material.
  • "Exogenous genetic material” is any genetic material, whether naturally occurring or otherwise, from any source that is capable of being inserted into any organism.
  • a construct or vector may include a promoter to express the protein or protein fragment of choice.
  • the promoter of the present invention is a hematopoietic stem cell-specific promoter.
  • Promoters that can be used in the present invention include the glucose-6-phosphotase promoter (Yoshiuchi et al, J. Clin. Endocrin. Metab. 53: 1016-1019 (1998), incorporated by reference in its entirety), interleukin- 1 alpha promoter (Mori and Prager, Leuk. Lymphoma 26:421- 433 (1997), incorporated by reference in its entirety), CMV promoter (Tong et al, Anticancer Res.
  • Lymphoma 25:415-425 (1997), incorporated by reference in its entirety), GM-CSF promoter (Shannon et al, Crit. Rev. Immunol. 77:301-323 (1997), incorporated by reference in its entirety), interleukin-5R alpha promoter (Sun et al, Curr. Top. Microbiol Immunol 277:173- 187 (1996), incorporated by reference in its entirety), interleukin-2 promoter (Serfing et al, Biochim. Biophys. Ada 7263: 181-200 ( 1995), incorporated by reference in its entirety; O'Neill et al, Transplant Proc.
  • Promoters suitable for expression of the GFAT II protein or fragment thereof of the present invention in bacteria have been described by Hawley and McClure, Nucleic Acids Res. 77:2237-2255 (1983), and Harley and Reynolds, Nucleic Acids Res. 75:2343-2361 (1987), both of which are incorporated by reference in their entirety.
  • Such promoters include, for example, the recA promoter (Fernandez de Henestrosa et al, FEMS Microbiol. Lett. 747:209-213 (1997); Nussbaumer et al, FEMS Microbiol. Lett.
  • Constructs or vectors may also include with the coding region of interest a nucleic acid sequence that acts, in whole or in part, to terminate transcription of that region.
  • Translational enhancers may also be incorporated as part of the vector DNA.
  • DNA constructs could contain one or more 5' non-translated leader sequences that may serve to enhance expression of the gene products from the resulting mRNA transcripts.
  • sequences may be derived from the promoter selected to express the gene or can be specifically modified to increase translation of the mRNA.
  • regions may also be obtained from viral RNAs, from suitable eukaryotic genes, or from a synthetic gene sequence.
  • a vector or construct may also include a screenable marker.
  • Screenable markers may be used to monitor expression.
  • Exemplary screenable markers include ⁇ -glucuronidase encoded by the uidA gene (GUS) (Jefferson, Plant Mol. Biol Rep. 5: 387-405 (1987); Jefferson et al, EMBO J. 6: 3901-3907 (1987)); ⁇ -lactamase (Sutcliffe et al, Proc. Natl. Acad. Sci.
  • luciferase Clontech, Palo Alto, CA, USA
  • ⁇ - galactosidase Clontech, Palo Alto, CA, USA
  • GST GST
  • Protein A Calbiochem
  • blue fluorescent protein Clontech, Palo Alto, CA, USA
  • fluorescent green protein Clontech, Palo Alto, CA, USA
  • selectable or screenable marker genes are also genes which encode a secretable marker whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers which encode a secretable antigen that can be identified by antibody interaction, or even secretable enzymes which can be detected by catalytic reactions.
  • Secretable proteins fall into a number of classes, including small, diffusible proteins which are detectable, (e.g., by ELISA), small active enzymes which are detectable in extracellular solution (e.g., ⁇ -amylase, ⁇ -lactamase, phosphinothricin transferase), or proteins which are inserted or trapped in the cell membrane (such as proteins which include a leader sequence).
  • Other possible selectable and/or screenable marker genes are apparent to those of skill in the art.
  • Suitable cells or cell lines may be bacterial cells.
  • E. coli are well-known as host cells in the field of biotechnology. Examples of such strains include E. coli strains JM101 (Yanish- Perron et al. Gene 33: 103-1 19 (1985), incorporated by reference in its entirety) and MON105 (Obukowicz et al, Applied Environmental Microbiology 55: 1511-1523 (1992), incorporated by reference in its entirety).
  • Also included in the present invention is the expression of the GFAT II protein or fragment thereof utilizing a chromosomal expression vector for E.
  • the gene encoding the GFAT II protein or fragment thereof of the present invention may also be constructed such that the 5' end of the gene codons are added to encode Met "2 -Ala "1 - or Met " ' at the N- terminus of the protein.
  • the N termini of proteins made in the cytoplasm of E. coli are affected by post-translational processing by methionine aminopeptidase (Bassat et al, J. Bac. 769:751-757 (1987), incorporated by reference in its entirety) and possibly by other peptidases so that upon expression the methionine is cleaved off the N- terminus.
  • the GFAT II protein of the present invention may be GFAT II polypeptides having Met "1 , Ala "1 or Met "2 -Ala "1 at the N-terminus. These GFAT II polypeptides may also be expressed in E. coli by fusing a secretion signal peptide of the N- terminus. This signal peptide can be cleaved from the polypeptide as part of the secretion process.
  • the GFAT II protein or fragment thereof of the present invention is expressed in a yeast cell, preferably Saccharomyces cerevisiae.
  • the GFAT II protein or fragment thereof of the present invention can be expressed in S. cerevisiae by fusing it to the N-terminus of the URA3, CYC1 or ARG3 genes (Guarente and Ptashne, Proc. Natl. Acad. Sci. (U.S.A.) 75:2199-2203 (1981); Rose et al, Proc. Natl Acad. Sci. (U.S.A.) 75:2460-2464 (1981); and Crabeel et al, EMBO J.
  • the GFAT II protein or fragment thereof of the present invention can be fused to either the PGK or TRP1 genes (Tuite et al, EMBO J. 7:603-608 (1982); and Dobson et al, Nucleic Acids. Res. 77:2287-2302 (1983), both of which are incorporated by reference in their entirety).
  • the GFAT II protein or fragment thereof of the present invention is expressed as a mature protein (Hitzeman et al, Nature 293:717-722 (1981); Valenzuela et al, Nature 295:347-350 (1982); and Derynck et al, Nucleic Acids Res. 77: 1819-1837 (1983), all of which are incorporated by reference in their entirety).
  • yeast promoters suitable for use in the present invention have been reviewed by Romanos et al, Yeast 5:423-488 (1992), incorporated by reference in its entirety.
  • the GFAT II protein or fragment thereof of the present invention is secreted by the yeast cell (Blobel and Dobberstein, J. Cell Biol.
  • insect cells may be utilized as host cells in the method of the present invention. See, e.g., V. A. Luckow, Protein Eng. J. L. Cleland., Wiley -Liss, New York, NY: 183-2180 (1996) and references cited therein.
  • general methods for expression of foreign genes in insect cells using baculovirus vectors are described in: O'Reilly et al, Baculovirus Expression Vectors: A Laboratory Manual. New York, W.H. Freeman and Company (1992), and King and Possee, 77 ⁇ e Baculovirus Expression System: A Laboratory Guide, London, Chapman & Hall, both of which are incorporated by reference in their entirety.
  • An expression vector is constructed comprising a baculovirus transfer vector, in which a strong baculovirus promoter (such as the polyhedrin promoter) drives transcription of a eukaryotic secretion signal peptide coding region, which is translationally joined to the coding region for the multi-functional protein.
  • a strong baculovirus promoter such as the polyhedrin promoter
  • the plasmid pVL1393 can be used.
  • two micrograms of this DNA is co-transfected with one microgram of baculovirus DNA into Spodoptera frugiperda insect cells, strain Sf9.
  • recombinant baculoviruses can be created using a baculovirus shuttle vector system (Luckow et al., J. Virol. 67: 4566- 4579 (1993), incorporated by reference in its entirety), now marketed as the Bac-To- BacTM Expression System (Life Technologies, Inc. Rockville, MD). Pure recombinant baculovirus carrying the multi-functional protein is used to infect cells cultured, for example, in Excell 401 serum- free medium (JRH Biosciences, Lenexa, Kansas) or Sf900-II (Life Technologies, Inc.). The multi-functional protein secreted into the medium can be recovered by standard biochemical approaches. Supernatants from mammalian or insect cells expressing the multi-functional proteins can be first concentrated using any of a number of commercial concentration units.
  • mammalian cells can be used to express the nucleic acid molecules of the present invention.
  • the nucleic acid molecules of the present invention are cloned into a suitable retroviral vector (see, e.g., Dunbar et al, Blood 55:3048-3057 (1995), herein incorporated by reference in its entirety; Baum et al, J. Hematother. 5: 323-329 (1996), incorporated by reference in its entirety; Bregni et al, Blood 80: 1418-1422 (1992), herein incorporated by reference in its entirety; Boris-Lawrie and Temin, Curr. Opin. Genet. Dev.
  • Transformation can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see for example Potrykus et al, Mol. Gen. Genet. 205:193-200 (1986); Lorz et al, Mol. Gen. Genet. 799:178 (1985); Fromm et al, Nature 379:791 (1986); Uchimiya et al, Mol Gen. Genet. 204:204 (1986); Marcotte et al, Nature 335:454-457 (1988), all of which are inco ⁇ orated by reference in their entirety).
  • the GFAT II molecules of the present invention are used to determine whether an individual has a mutation affecting the level (i.e., the concentration of GFAT II mRNA or protein in a sample, etc.) or pattern (i.e., the kinetics of expression, rate of decomposition, stability profile, etc.) of the GFAT II expression (collectively, the "GFAT II Response" of a cell or bodily fluid) (for example, a mutation in the GFAT II gene, or in a regulatory region(s) or other gene(s) that control or affect the expression of GFAT II), and being predictive of individuals who would be predisposed to, for example NIDDIM, and other disorders.
  • a mutation affecting the level i.e., the concentration of GFAT II mRNA or protein in a sample, etc.
  • pattern i.e., the kinetics of expression, rate of decomposition, stability profile, etc.
  • the GFAT II Response for example, a mutation in the GFAT II gene, or
  • the GFAT II Response manifested by a cell or bodily fluid is said to be "altered” if it differs from the GFAT II Response of cells or of bodily fluids of normal individuals. Such alteration may be manifested by either abnormally increased or abnormally diminished GFAT II Response.
  • the GFAT II Response manifested by the cell or bodily fluid of the patient is compared with that of a similar cell sample (or bodily fluid sample) of normal individuals.
  • such an analysis is conducted by determining the presence and/or identity of polymo ⁇ hism(s) in the GFAT II gene or its flanking regions which are associated with a disorder.
  • the GFAT II cDNA sequence (or a sub-sequence thereof) may be employed as a marker nucleic acid molecule to identify such polymo ⁇ hism(s).
  • such polymorphisms can be detected through the use of a marker nucleic acid molecule or a marker protein that is genetically linked to (i.e., a polynucleotide that co-segregates with) such polymo ⁇ hism(s).
  • a marker nucleic acid molecule or a marker protein that is genetically linked to (i.e., a polynucleotide that co-segregates with) such polymo ⁇ hism(s).
  • the GFAT II gene and/or a sequence or sequences that specifically hybridize to the GFAT II gene have been mapped.
  • GFAT II gene and/or a sequence or sequences that specifically hybridize to the GFAT II gene have been mapped to chromosome 5, q33.5-37.5, and preferably mapped to chromosome 5, q34.5-36.5 and even more preferably chromosome 5, q35.5.
  • marker nucleic acid molecules will have the nucleotide sequence of a polynucleotide that is closely genetically linked to such polymo ⁇ hism(s). Polynucleotide markers that map to such locations are well known and can be employed to identify such polymo ⁇ hism(s).
  • such polymo ⁇ hisms can be detected through the use of a marker nucleic acid molecule that is physically linked to such polymo ⁇ hism(s).
  • marker nucleic acid molecules comprising a nucleotide sequence of a polynucleotide located within 1 mb of the polymo ⁇ hism(s), and more preferably within 100 kb of the polymo ⁇ hism(s), and most preferably within 10 kb of the polymo ⁇ hism(s) can be employed.
  • a "polymo ⁇ hism" in the GFAT II gene or its flanking regions is a variation or difference in the sequence of the GFAT II gene or its flanking regions that arises in some of the members of a species.
  • the variant sequence and the "original" sequence co-exist in the species' population. In some instances, such co-existence is in stable or quasi-stable equilibrium.
  • a polymo ⁇ hism is thus said to be "allelic,” in that, due to the existence of the polymorphism, some members of a species may have the original sequence (i.e. the original "allele") whereas other members may have the variant sequence (i.e. the variant "allele”). In the simplest case, only one variant sequence may exist, and the polymo ⁇ hism is thus said to be di-allelic. In other cases, the species' population may contain multiple alleles, and the polymo ⁇ hism is termed tri-allelic, etc. A single gene may have multiple different unrelated polymo ⁇ hisms.
  • the polymo ⁇ hism may have a di- allelic polymo ⁇ hism at one site, and a multi-allelic polymo ⁇ hism at another site.
  • the variation that defines the polymo ⁇ hism may range from a single nucleotide variation to the insertion or deletion of extended regions within a gene.
  • the DNA sequence variations are in regions of the genome that are characterized by short tandem repeats (STRs) that include tandem di- or tri-nucleotide repeated motifs of nucleotides.
  • STRs short tandem repeats
  • Polymo ⁇ hisms characterized by such tandem repeats are referred to as "variable number tandem repeat" (“VNTR") polymo ⁇ hisms.
  • VNTRs have been used in identity and paternity analysis (Weber, U.S.
  • the detection of polymo ⁇ hic sites in a sample of DNA may be facilitated through the use of nucleic acid amplification methods. Such methods specifically increase the concentration of polynucleotides that span the polymo ⁇ hic site, or include that site and sequences located either distally or proximally to it. Such amplified molecules can be readily detected by gel electrophoresis or other means.
  • PCR polymerase chain reaction
  • LCR Ligase Chain Reaction
  • LCR can be performed with oligonucleotides having the proximal and distal sequences of the same strand of a polymorphic site.
  • either oligonucleotide will be designed to include the actual polymo ⁇ hic site of the polymo ⁇ hism.
  • the reaction conditions are selected such that the oligonucleotides can be ligated together only if the target molecule either contains or lacks the specific nucleotide that is complementary to the polymo ⁇ hic site present on the oligonucleotide.
  • the oligonucleotides may be selected such that they do not include the polymo ⁇ hic site (see, Segev, PCT Application WO 90/01069).
  • Oligonucleotide Ligation Assay (“OLA”) may alternatively be employed
  • OLA oligonucleotide
  • nucleic acid amplification procedures such as allele-specific oligomers, branched DNA technology, transcription-based amplification systems, or isothermal amplification methods may also be used to amplify and analyze such polymo ⁇ hisms (Malek, et al., U.S. Patent 5,130,238; Davey, et al., European Patent Application 329,822; Schuster et al, U.S. Patent 5,169,766; Miller, et al, PCT appln. WO 89/06700; Kwoh, et al, Proc. Natl. Acad. Sci.
  • the identification of a polymo ⁇ hism in the GFAT II gene can be determined in a variety of ways. By correlating the presence or absence of NIDDIM in an individual with the presence or absence of a polymo ⁇ hism in the GFAT II gene or its flanking regions, it is possible to diagnose the predisposition of an asymptomatic patient to NIDDIM or other diseases. If a polymo ⁇ hism creates or destroys a restriction endonuclease cleavage site, or if it results in the loss or insertion of DNA (e.g., a VNTR polymo ⁇ hism), it will alter the size or profile of the DNA fragments that are generated by digestion with that restriction endonuclease.
  • RFLPs restriction fragment length polymo ⁇ hisms
  • a sample DNA is obtained from a patient's cells.
  • the DNA sample is obtained from the patient's blood.
  • any source of DNA may be used.
  • the DNA is subjected to restriction endonuclease digestion.
  • GFAT II is used as a probe in accordance with the above-described RFLP methods.
  • the polymo ⁇ hism obtained in this approach can then be cloned to identify the mutation at the coding region which alters the protein's structure, or at the regulatory region of the gene which affects its expression level. Changes involving promoter interactions with other regulatory proteins can be identified by, for example, gel shift. Several different classes of polymo ⁇ hisms may be identified through such methods.
  • Examples of such classes include: (1) polymo ⁇ hisms present in the GFAT II cDNA of different individuals; (2) polymo ⁇ hisms in non-translated GFAT II gene sequences, including the promoter or other regulatory regions of the GFAT II gene; (3) polymo ⁇ hisms in genes whose products interact with GFAT II regulatory sequences; (4) polymo ⁇ hisms in gene sequences whose products interact with the GFAT II protein, or to which the GFAT II protein binds.
  • the evaluation is conducted using oligonucleotide "probes" whose sequence is complementary to that of a portion of GFAT II mRNA. Such molecules are then incubated with cell extracts of a patient under conditions sufficient to permit nucleic acid hybridization. For this sub- embodiment, cells of the trabecular meshworks are preferred.
  • the detection of double-stranded probe-mRNA hybrid molecules is indicative of the presence of GFAT II mRNA; the amount of such hybrid formed is proportional to the amount of GFAT II mRNA.
  • probes may be used to ascertain the level and extent of GFAT II mRNA production in a patient's cells.
  • Such nucleic acid hybridization may be conducted under quantitative conditions (thereby providing a numerical value of the amount of GFAT II mRNA present).
  • the assay may be conducted as a qualitative assay that indicates either that GFAT II mRNA is present, or that its level exceeds a user set, predefined value.
  • the previously described "anti-GFAT II antibodies" are employed in an immunodiagnostic assay.
  • a biopsy or a macrophage or other blood cell sample
  • a sample of bodily fluid especially, blood, serum, plasma, tears, etc.
  • the anti-GFAT II antibodies of the present invention may thus be used in an immunoassay to assess the presence of GFAT II.
  • Any of a wide array of immunoassays formats may be used for this pu ⁇ ose (Fackrell, Clin. Immunoassay 5:213-219 (1985)), Yolken, Rev. Infect. Dis. 4:35 (1982); Collins, In: Alternative Immunoassays, John Wiley & Sons, NY (1985); Ngo, et al, In: Enzyme Mediated Immunoassay, Plenum Press, NY (1985), all of which are herein inco ⁇ orated by reference in their entirety).
  • the simplest immunoassay involves merely incubating an antibody that is capable of binding to a predetermined target molecule with a sample suspected to contain the target molecule.
  • the presence of the target molecule is determined by the presence, and proportional to the concentration, of any antibody bound to the target molecule.
  • a solid phase is typically employed.
  • the sample can be passively bound to a solid support, and, after incubation with the antibody, the support can be washed to remove any unbound antibody.
  • the concentration of the target molecule is determined by binding the antibody to a support, and then permitting the support to be in contact with a sample suspected of containing the target molecule.
  • Target molecules that have become bound to the immobilized antibody can be detected in any of a variety of ways.
  • the support can be incubated in the presence of a labeled, second antibody that is capable of binding to a second epitope of the target molecule. Immobilization of the labeled antibody on the support thus requires the presence of the target, and is proportional to the concentration of the target in the sample.
  • the target is incubated with the sample and with a known amount of labeled target. The presence of target molecule in the sample competes with the labeled target molecules for antibody binding sites.
  • the amount of labeled target molecules that are able to bind the antibody is inversely proportional to the concentration of target molecule in the sample.
  • RIAs radioactive labels
  • ELISAs enzyme labels
  • RIAs have the advantages of simplicity, sensitivity, and ease of use. Radioactive labels are of relatively small atomic dimension, and do not normally affect reaction kinetics. Such assays suffer, however, from the disadvantages that, due to radioisotopic decay, the reagents have a short shelf-life, require special handling and disposal, and entail the use of complex and expensive analytical equipment.
  • RIAs are described in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, et al, North Holland Publishing Company, NY (1978), with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques” by Chard, inco ⁇ orated by reference herein in its entirety.
  • ELISAs have the advantage that they can be conducted using inexpensive equipment, and with a myriad of different enzymes, such that a large number of detection strategies —colorimetric, pH, gas evolution, etc. — can be used to quantitate the assay.
  • the enzyme reagents have relatively long shelf-lives, and lack the risk of radiation contamination that attends to RIA use.
  • ELISAs are described in ELISA and Other Solid Phase Immunoassays (Kemeny, et al., Eds.), John Wiley & Sons, NY (1988), and Using Antibodies: A Laboratory Manual, (Harlow and Lane, Eds.) CSH Press (1998).
  • Anti-GFAT II antibodies or GFAT II binding molecules may be administered to a patient, and their capacity to bind to GFAT II in vivo may be determined by ocular examination. Significantly, since such a diagnostic test is relatively rapid, immune responses that require significant time, such as the potential eliciting of anti-[anti- GFAT II] antibodies, or the complexing of such antibodies with anti-GFAT II antibodies, is not important.
  • the antibody will be fluorescently labeled, and will be provided to a patient by injection into the patient's circulatory system.
  • a GFAT II protein or fragment thereof can be used in assays for screening test substances for the ability to modulate or maintain GFAT II activity.
  • the test substance is an agonist, antagonist, or small molecule inhibitor of the GFAT II protein.
  • the test substance may bind to GFAT II substrate.
  • the test substance may also be an agonist, antagonist, or small molecule inhibitor of GFAT I.
  • Assays for screening GFAT II activity include, for example, a colorimetric assay (Kaufman et al., Enzyme 12:537 (1971), Richards and Greengard, Biochimica et Biophysica Acta 304:842-850 (1973), McKnight et al, J. Biochem. 267:25208-25212 (1992), inco ⁇ orated by reference in their entireties) or a HPLC-based separation technique (Buse et al. Am J. Physiol. 272: E1080-1088 (1997), Nelson et al, Am. J. Physiol 272: E848-855 (1997), inco ⁇ orated by reference in their entireties).
  • a colorimetric assay Kerman et al., Enzyme 12:537 (1971), Richards and Greengard, Biochimica et Biophysica Acta 304:842-850 (1973), McKnight et al, J. Biochem. 267:25208-25212 (1992), inco
  • the agents of the present invention can be formulated according to known methods to prepare pharmacologically acceptable compositions, whereby these materials, or their functional derivatives, having the desired degree of purity, are combined in admixture with a physiologically acceptable carrier, excipient, or stabilizer. Such materials are non-toxic to recipients at the dosages and concentrations employed.
  • the active component of such compositions may be GFAT II protein, GFAT II fusion proteins or fragments of GFAT II protein or analogs or mimetics of such molecules. Where nucleic acid molecules are employed, such molecules may be sense, antisense or triplex oligonucleotides of the GFAT II cDNA or gene.
  • a composition is said to be "pharmacologically acceptable” if its administration can be tolerated by a recipient patient.
  • An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.
  • Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences (16th ed., Osol, Ed., Mack, Easton PA (1980), herein inco ⁇ orated by reference in its entirety).
  • the composition is to be water soluble, it may be formulated in a buffer such as phosphate or other organic acid salt preferably at a pH of about 7 to 8.
  • the composition is only partially soluble in water, it may be prepared as a microemulsion by formulating it with a nonionic surfactant such as Tween, Pluronics, or PEG, e.g., Tween 80, in an amount of, for example, 0.04-0.05% (w/v), to increase its solubility.
  • a nonionic surfactant such as Tween, Pluronics, or PEG, e.g., Tween 80
  • water soluble as applied to the polysaccharides and polyethylene glycols is meant to include colloidal solutions and dispersions.
  • the solubility of the cellulose derivatives is determined by the degree of substitution of ether groups, and the stabilizing derivatives useful herein should have a sufficient quantity of such ether groups per anhydroglucose unit in the cellulose chain to render the derivatives water soluble.
  • a degree of ether substitution of at least 0.35 ether groups per anhydroglucose unit is generally sufficient.
  • antioxidants e.g., ascorbic acid
  • low molecular weight polypeptides e.g., polyarginine or tripeptides
  • proteins such as serum albumin, gelatin, or immunoglobulins
  • hydrophilic polymers such as polyvinyl pyrrolidone
  • amino acids such as glycine, glutamic acid, aspartic acid, or arginine
  • monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins
  • chelating agents such as EDTA
  • sugar alcohols such as mannitol or sorbitol.
  • Controlled or sustained release preparations may be achieved through the use of polymers to complex or absorb the GFAT II molecule(s) of the composition.
  • the controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the methods of inco ⁇ oration in order to control release.
  • Sustained release formulations may also be prepared, and include the formation of microcapsular particles and implantable articles.
  • the GFAT II molecule(s) of the composition is preferably inco ⁇ orated into a biodegradable matrix or microcapsule.
  • a suitable material for this pu ⁇ ose is a polylactide, although other polymers of poly-(a- hydroxycarboxylic acids), such as poly-D-(-)-3-hydroxybutyric acid (EP 133,988A), can be used.
  • Other biodegradable polymers include poly(lactones), poly(orthoesters), polyamino acids, hydrogels, or poly(orthocarbonates) poly(acetals).
  • the polymeric material may also comprise polyesters, polyQactic acid) or ethylene vinylacetate copolymers.
  • sustained release compositions see U.S. Patent No. 3,773,919, EP 58,481A, U.S. Patent No. 3,887,699, EP 158,277A, Canadian Patent No. 1176565, Sidman, U. et al, Biopolymers 22:547 (1983), and Langer, R. et al, Chem. Tech. 72:98 (1982).
  • microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatine-microcapsules and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • liposome formulations and methods that permit intracellular uptake of the molecule will be employed. Suitable methods are known in the art; see, for example, Chicz, et al. PCT Application WO 94/04557, Jaysena, et al. (PCT Application WO93/ 12234, Yarosh, U.S. Patent No. 5,190,762, Callahan, et al. U.S. Patent No. 5,270,052 and Gonzalezro, PCT Application 91/05771, all of which are herein inco ⁇ orated by reference.
  • compositions of the present invention may be sterilized, as by filtration through sterile filtration membranes (e.g., 0.2 micron membranes).
  • sterile filtration membranes e.g., 0.2 micron membranes
  • the compositions may be stored in lyophilized form or as a liquid solution. It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of salts of the molecules.
  • Example 1 DNA from a GFAT II clone is labeled with digoxigen dUTP by nick translation.
  • Labeled probe is combined with sheared human DNA and hybridized to normal metaphase chromosomes derived from PHA stimulated peripheral blood lymphocytes in a solution containing 50% formamie, 10% dextran sulphate, and 2 X SSC.
  • Specific hybridization signals are detected by incubating the hybridized slides in floreseceinated antidigoxigenin antibodies followed by counterstaining with DAPI for one color experiments.
  • Probe detection for two color experiments is accomplished by incubating the slides in fluoresceniated antidigoxigenin antibodies and Texas red avidin followed by counterstaining with DAPI.
  • RNA dot blots of human tissues are carried out essentially according to the manufacturers instructions (Product No: 7770-1, Clontech, Palo Alto, CA, USA). An RNA dot blot containing normalized loadings of 89-514 ng of each poly A + RNA per dot from different human tissues and different controls are probed with []. Results are set forth in Table 1.
  • RNA Northern blots of human tissues are carried out essentially according to the manufacturer's instructions (Clontech, Palo Alto, CA, USA). Northern blots containing approximately 2 ⁇ g of poly A + RNA per lane from different tissues are prepared (Product No; 7760-1, Clontech, Palo Alto, CA, USA). Results are set forth in Table 2.
  • rhGFAT I Glutamine:Fructose-6-phosphate AmidoTransferase
  • RhGFAT Glutamine:Fructose-6-phosphate AmidoTransferase
  • RhGFAT is overexpressed in insect cells using a baculovirus infection vector. Enzyme activity is identified in the cytosolic fraction and was purified partially by chromatography on DEAE-Sepharose. Identification of test substances is performed in an assay volume of 50 ml in a 96 well format.
  • Enzyme (rhGFAT I) is added to initiate the assay containing 20 mM Imidazole pH 6.8, 1 mg/ml BSA, 0.4 mM DTT, 10% Glycerol, 10 mM KC1, 20 mM l4 C-Fructose-6-phosphate and 400 mM L- Glutamine. After a 60 min incubation, the assay is stopped by adding 150 ml of a suspension of Dowex AG1X8 anion exchange resin equilibrated in 10 mM sodium formate buffer pH 3.0. Unreacted C-Fructose-6-phosphate is captured by the resin, whereas l4 C-Glucosamine-6-phosphate is unbound and remains in the buffer.
  • the product is quantified by removing a 50 ml aliquot, adding 200 ml of scintillation cocktail and counting in a Packard Topcount.
  • GFAT II activity is measured by separating GFAT II substrate from its product.

Abstract

La présente invention a trait à un ADNc de rat, de souris et d'homme codant pour une glutamine: une enzyme de type II, la fructose-6-phosphate aminotransférase. Cette invention a également trait à des molécules d'acide nucléique associées à un ADNc ou qui en sont dérivées, comprenant leurs compléments, leurs homologues et leurs fragments. L'invention concerne aussi des procédés d'utilisation de ces molécules d'acide nucléique, par exemple, pour produire une protéine et ses fragments et pour rechercher par criblage des composés ou des compositions qui spécifiquement ou de façon préférentielle réalise l'activité d'une protéine GFAT II.
PCT/US1999/030943 1998-12-22 1999-12-22 Glutamine: une enzyme de type ii, la fructose-6-phosphate aminotransferase, et ses acides nucleiques codants WO2000037617A1 (fr)

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WO2001096574A1 (fr) * 2000-06-13 2001-12-20 Otsuka Pharmaceutical Co., Ltd. Nouveau gene enzymatique et son produit d'expression
WO2002099083A2 (fr) 2001-06-05 2002-12-12 Exelixis, Inc. Gfat en tant que modificateurs de la voie p53 et leur procede d'utilisation
WO2003023063A1 (fr) * 2001-09-07 2003-03-20 Sankyo Company, Limited Methode d'estimation du risque d'apparition de diabetes
JP2009511004A (ja) * 2005-10-05 2009-03-19 バイエル・クロップサイエンス・アーゲー 増加したアミノ糖含量を有する植物

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WO1993021330A1 (fr) * 1992-04-22 1993-10-28 Zymogenetics, Inc. Glutamine:fluctose-6-phosphate amidotransferase humaine

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001096574A1 (fr) * 2000-06-13 2001-12-20 Otsuka Pharmaceutical Co., Ltd. Nouveau gene enzymatique et son produit d'expression
JP2002065284A (ja) * 2000-06-13 2002-03-05 Otsuka Pharmaceut Co Ltd 新規酵素遺伝子およびその発現産物
US7091018B2 (en) 2000-06-13 2006-08-15 Otsuka Pharmaceutical Co., Ltd. Enzyme gene and its expression product
WO2002099083A2 (fr) 2001-06-05 2002-12-12 Exelixis, Inc. Gfat en tant que modificateurs de la voie p53 et leur procede d'utilisation
EP1456650A2 (fr) * 2001-06-05 2004-09-15 Exelixis, Inc. Gfat en tant que modificateurs de la voie p53 et leur procede d'utilisation
EP1456650A4 (fr) * 2001-06-05 2005-11-02 Exelixis Inc Gfat en tant que modificateurs de la voie p53 et leur procede d'utilisation
WO2003023063A1 (fr) * 2001-09-07 2003-03-20 Sankyo Company, Limited Methode d'estimation du risque d'apparition de diabetes
JP2009511004A (ja) * 2005-10-05 2009-03-19 バイエル・クロップサイエンス・アーゲー 増加したアミノ糖含量を有する植物

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