WO2000014251A2 - Transferases humaines - Google Patents

Transferases humaines Download PDF

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Publication number
WO2000014251A2
WO2000014251A2 PCT/US1999/020989 US9920989W WO0014251A2 WO 2000014251 A2 WO2000014251 A2 WO 2000014251A2 US 9920989 W US9920989 W US 9920989W WO 0014251 A2 WO0014251 A2 WO 0014251A2
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WIPO (PCT)
Prior art keywords
seq
trnsfs
polynucleotide
sequence
sequences
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PCT/US1999/020989
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English (en)
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WO2000014251A3 (fr
Inventor
Y. Tom Tang
Neil C. Corley
Karl J. Guegler
Mariah R. Baughn
Preeti Lal
Henry Yue
Jennifer L. Hillman
Yalda Azimzai
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Incyte Pharmaceuticals, Inc.
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Application filed by Incyte Pharmaceuticals, Inc. filed Critical Incyte Pharmaceuticals, Inc.
Priority to AU61428/99A priority Critical patent/AU6142899A/en
Priority to EP99948200A priority patent/EP1109917A2/fr
Priority to JP2000568992A priority patent/JP2004512804A/ja
Priority to US09/786,240 priority patent/US6558935B1/en
Priority to CA002343351A priority patent/CA2343351A1/fr
Publication of WO2000014251A2 publication Critical patent/WO2000014251A2/fr
Publication of WO2000014251A3 publication Critical patent/WO2000014251A3/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.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • methyl transferases transfer one-carbon methyl groups
  • amino transferases transfer nitrogenous amino groups
  • similarly denominated enzymes transfer aldehyde or ketone, acyl, glycosyl, alkyl or aryl, isoprenyl, saccharyl, phosphorous-containing, sulfur-containing, or selenium-containing groups, as well as small enzymatic groups such as Coenzyme A.
  • HGPRT HGPRT-Nyhan syndrome
  • PAPS 3'-phosphoadenosine-5'-phosphosulfate
  • ATP adenosine triphosphate
  • APS adenosine 5'-phosphosulfate
  • ATP sulfurylase catalyzes the formation of APS from ATP and free sulfate.
  • APS kinase phosphorylates APS to produce PAPS, the sole source of donor sulfate in higher organisms. In bacteria, fungi, yeast, and plants, these two enzymes are separate polypeptides. In animals, ATP sulfurylase and APS kinase are present in a single protein.
  • phosphatidylethanolamine ⁇ -methyltransferase catalyzes the methylation of phosphatidylethanolamine.
  • Hepatocytes in the liver synthesize phosphatidylcholine (PC) by stepwise methylation of phosphatidylethanolamine and have abundant activity for PEMT.
  • PC phosphatidylcholine
  • Other cells and tissues express minimal activities for PEMT. All mammalian cells, including hepatocytes, synthesize PC from choline via the CDP-choline pathway.
  • H ⁇ K-1 sulfotransferase forms the HNK-1 carbohydrate epitope by adding a sulfate group to glycoproteins and glycolipids.
  • the HNK-1 epitope was discovered by an antibody against human natural killer cells and is found in neural adhesion molecules, including N-CAM and myelin-associate glycoprotein.
  • the HNK-1 carbohydrate epitope was recognized to have functional significance as an auto-antigen involved in peripheral demyelinative neuropathy.
  • the HNK-1 ST is a type II membrane protein with a consensus sequence shared by Golgi- associated sulfotransferases.
  • the human and rat HNK- 1 STs share 90% homology in amino acid sequence.
  • the enzyme glycine N-methyltransferase catalyzes the transfer of the methyl group from S-adenosylmethionine to glycine to form S-adenosylhomocyteine and sarcosine.
  • Glycine N- 5 methyltransferase is a tetramer of identical subunits, has a nucloetide binding region, and is localized in the liver. Amino acid sequence homology is found between glycine N- methlytransferases from rat, rabbit, pig, and human livers.
  • Glycine N-methyltransferase can exist as a dimer which binds polycyclic aromatic hydrocarbons (PAHs) and acts as a transcriptional activator (Ogawa, H. et al. (1998) Int. J. Biochem. Cell Biol. 30:13-26; Bhat, R. and Bresnick, E.
  • PAHs polycyclic aromatic hydrocarbons
  • N-acylation with the 14-carbon fatty acid, myristate is found on the amino groups of N- terminal glycines of a number of proteins that are essential to normal cell functioning and/or are potential therapeutic targets of disease.
  • proteins include subunits of
  • N-myristoylated proteins are associated with a variety of organelles with the myristate moiety required for such diverse functions as specific protein-protein or protein-lipid interactions, ligand-induced protein conformational changes, and correct subcellular targeting.
  • Protein myristoylation occurs almost exclusively cotranslationally during protein synthesis of the first 100 amino acids.
  • the reaction is catalyzed by the enzyme myristoyl CoA:protein N-
  • NMT myristoyl-transferase 1 (EC 2.3.1.97).
  • Immunofluorescence microscopy reveals NMT to be distributed uniformly throughout the cytoplasm of yeast and mammalian cells. This finding, plus evidence that N- myristoylation occurs on nascent polypeptides bound to free polyribosomes, establish that NMT is physically localized and functionally active in the cell cytoplasm.
  • Protein N-myristoylation appears to be a tightly regulated process involving i) the coordinated participation of several different enzymes/proteins, e.g. N-methionylaminopeptidase, fatty acid synthetase, long chain acyl-CoA synthetase, acyl-CoA-binding proteins; ii) access of NMT to pools of myristoyl-CoA substrate; and iii) N-myristoylation of nascent polypeptide substrates during protein synthesis to avoid potential interfering reactions such as N-acetylation and polypeptide folding.
  • enzymes/proteins e.g. N-methionylaminopeptidase, fatty acid synthetase, long chain acyl-CoA synthetase, acyl-CoA-binding proteins.
  • NMT The ability of NMT to function in regulated N-myristoylation has implied the existence of mechanisms designed to ensure targeting of NMT to the appropriate protein synthesis machinery. These mechanisms may involve interactions with other cooperating components that facilitate the recognition and efficient N-myristoylation of the rapidly growing polypeptide substrates.
  • Protein N-myristoylation activity may be a chemotherapeutic target for cancer, infectious diseases, and immune disorders. Antagonists of NMT may reduce posttranslational myristoylation of oncoproteins and other growth-activating cellular proteins.
  • glycosylated proteins that have covalently attached carbohydrate chains, or oligosaccharides. Some of these glycoproteins have only one or a few carbohydrate groups while others have numerous oligosaccharide side chains, which may be linear or branched.
  • the sugar residues of many plasma membrane glycoproteins orient these proteins in membranes.
  • Sugar residues of glycoproteins are hydrophilic and strongly prefer to be located near the aqueous or extracellular surface rather than the hydrocarbon core of the plasma membrane. Because there is a high energy barrier to the rotation of a glycoprotein from one side of the membrane to the other, the carbohydrate groups of membrane glycoproteins help to maintain the asymmetric character of biological membranes.
  • glycophorin a protein found in the membrane of red blood cells. Many soluble glycoproteins are known as well, including carrier proteins, antibodies, and many of the proteins contained in lysosomes. Carbohydrate groups of plasma membrane glycoproteins play a major role in cell-cell recognition. Oligosaccharides are involved in many inflammatory processes and may also provide targets for tumor immunotherapy.
  • Glycoproteins are often linked to their oligosaccharides through asparagine (N) residues. These N-linked oligosaccharides are very diverse, but the many pathways by which they all form have a common first step.
  • ADP-glucose pyrophosphorylases are tetrameric, allosterically regulated enzymes. There are a number of conserved regions in the sequence of bacterial and plant ADP-glucose pyrophosphorylase subunits. Additionally, there are three regions which are considered signature patterns (ExPASy PROSITE database, documents PS00808-PS00810). The first two regions are N-terminal and have been proposed to be part of the allosteric and substrate-binding sites in the Escherichia coli enzyme. The third pattern corresponds to a conserved region in the central part of the enzymes. In eukaryotic cells, mannose- 1 -phosphate guanyltransferase is involved in early steps of protein glycosylation.
  • Mannose- 1 -phosphate guanyltransferase (MPG), also referred to as NDP-hexose pyrophosphorylase or GDP-mannose pyrophosphorylase B, catalyzes the conversion of GTP and ⁇ -D-mannose 1 -phosphate into diphosphate and CDP- ethanolamine.
  • MPG Mannose- 1 -phosphate guanyltransferase
  • NDP-hexose pyrophosphorylase also referred to as NDP-hexose pyrophosphorylase or GDP-mannose pyrophosphorylase B
  • This enzyme is very similar to CDP-glucose pyrophosphorylase and may be involved in the regulation of cell cycle progression.
  • a cDNA coding for MPG1 was recently isolated from a Trichoderma reesei cDNA library (Kruszewska, J. S. et al.
  • CDGS type IB A new type of CDGS, designated as CDGS type IB, has recently been described (Niehues, R. et al. (1998) Clin. Invest. 101:1414-1420). The clinical phenotype of this new disorder is fundamentally different from other types of CDGS in that no psychomotor or mental retardation is present.
  • the invention features substantially purified polypeptides, human transferase proteins, referred to collectively as "TRNSFS” and individually as “TRNSFS- 1,” “TRNSFS-2,” “TRNSFS- 3,” “TRNSFS-4,” “TRNSFS-5,” “TRNSFS-6,” “TRNSFS-7,” “TRNSFS-8,” “TRNSFS-9,” “TRNSFS-10,” “TRNSFS-11,” “TRNSFS-12,” “TRNSFS-13,” “TRNSFS-14,” and “TRNSFS- 15.”
  • the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:
  • the invention further provides a substantially purified variant having at least 90% amino acid identity to at least one of the amino acid sequences selected from the group consisting of SEQ ID NO: 1-15 and fragments thereof.
  • the invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15 and fragments thereof.
  • the invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15 and fragments thereof.
  • the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15 and fragments thereof.
  • the invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1-15 and fragments thereof.
  • the invention also provides a method for detecting a polynucleotide in a sample containing nucleic acids, the method comprising the steps of (a) hybridizing the complement of the polynucleotide sequence to at least one of the polynucleotides of the sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide in the sample.
  • the method further comprises amplifying the polynucleotide prior to hybridization.
  • the invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 (SEQ ID NO: 16-30), and fragments thereof.
  • the invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide sequence selected from the group consisting of SEQ ID NO: 16-30 and fragments thereof.
  • the invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 16-30 and fragments thereof.
  • the invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15 and fragments thereof.
  • the expression vector is contained within a host cell.
  • the invention also provides a method for producing a polypeptide, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
  • the invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 1-15 and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
  • the invention further includes a purified antibody which binds to a polypeptide selected from the group consisting of SEQ ID NO: 1-15 and fragments thereof.
  • the invention also provides a purified agonist and a purified antagonist to the polypeptide.
  • the invention also provides a method for treating or preventing a disorder associated with decreased expression or activity of TRNSFS, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 1-15 and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
  • the invention also provides a method for treating or preventing a disorder associated with increased expression or activity of TRNSFS, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15 and fragments thereof.
  • Figures 2A, 2B, 2C and 2D show the amino acid sequence alignments among TRNSFS-2 (2682663; SEQ ID NO:2), Arabidopsis thaliana MPG (GI 2642159; SEQ ID NO:32), and
  • Caenorhabditis elegans MPG (GI 2804432; SEQ ID NO:33), produced using the multisequence alignment program of LASERGENE software (DNASTAR Inc, Madison WI).
  • Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ ID NO), clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments used to assemble full-length sequences encoding TRNSFS.
  • Table 2 shows features of each polypeptide sequence, including potential motifs, homologous sequences, and methods and algorithms used for identification of TRNSFS.
  • Table 3 shows useful fragments of each nucleic acid sequence; the tissue-specific expression patterns of each nucleic acid sequence as determined by northern analysis; diseases, disorders, or conditions associated with these tissues; and the vector into which each cDNA was cloned.
  • Table 4 describes the tissues used to construct the cDNA libraries from which cDNA clones encoding TRNSFS were isolated.
  • Table 5 shows the tools, programs, and algorithms used to analyze TRNSFS, along with applicable descriptions, references, and threshold parameters.
  • TRNSFS refers to the amino acid sequences of substantially purified TRNSFS obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and preferably the human species, from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • altered nucleic acid sequences encoding TRNSFS include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide the same as TRNSFS or a polypeptide with at least one functional characteristic of TRNSFS. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding TRNSFS, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding TRNSFS.
  • amino acid and amino acid sequence refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules.
  • fragments refer to fragments of TRNSFS which are preferably at least 5 to about 15 amino acids in length, most preferably at least 14 amino acids, and which retain some biological activity or immunological activity of TRNSFS.
  • amino acid sequence is recited to refer to an amino acid sequence of a naturally occurring protein molecule
  • amino acid sequence and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • antisense refers to any composition containing a nucleic acid sequence which is complementary to the "sense” strand of a specific nucleic acid sequence. Antisense molecules may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and to block either transcription or translation. The designation “negative” can refer to the antisense strand, and the designation “positive” can refer to the sense strand.
  • biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
  • immunologically active refers to the capability of the natural, recombinant, or synthetic TRNSFS, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • complementarity refers to the natural binding of polynucleotides by base pairing.
  • sequence 5' A-G-T 3'
  • complementary sequence 3' T-C-A 5'.
  • Complementarity between two single-stranded molecules may be “partial,” such that only some of the nucleic acids bind, or it may be “complete,” such that total complementarity exists between the single stranded molecules.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands, and in the design and use of peptide nucleic acid (PNA) molecules.
  • PNA peptide nucleic acid
  • composition comprising a given polynucleotide sequence and a “composition comprising a given amino acid sequence” refer broadly to any composition containing the given polynucleotide or amino acid sequence.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotide sequences encoding TRNSFS or fragments of TRNSFS may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
  • salts e.g., NaCl
  • detergents e.g., sodium dodecyl sulfate; SDS
  • other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • Consensus sequence refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, extended using the XL-PCR kit (Perkin-Elmer, Norwalk CT) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from the overlapping sequences of more than one Incyte Clone using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison WI). Some sequences have been both extended and assembled to produce the consensus sequence.
  • correlates with expression of a polynucleotide indicates that the detection of the presence of nucleic acids, the same or related to a nucleic acid sequence encoding TRNSFS, by northern analysis is indicative of the presence of nucleic acids encoding TRNSFS in a sample, and thereby correlates with expression of the transcript from the polynucleotide encoding TRNSFS.
  • derivative refers to the chemical modification of a polypeptide sequence, or a polynucleotide sequence. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • similarity refers to a degree of complementarity. There may be partial similarity or complete similarity.
  • the word "identity” may substitute for the word “similarity.”
  • a partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as “substantially similar.”
  • the inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization, and the like) under conditions of reduced stringency.
  • a substantially similar sequence or hybridization probe will compete for and inhibit the binding of a completely similar (identical) sequence to the target sequence under conditions of reduced stringency. This is not to say that conditions of reduced stringency are such that non-specific binding is permitted, as reduced stringency conditions require that the binding of two sequences to one another be a specific (i.e., a selective) interaction.
  • the absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% similarity or identity). In the absence of non-specific binding, the substantially similar sequence or probe will not hybridize to the second non-complementary target sequence.
  • a partial degree of complementarity e.g., less than about 30% similarity or identity
  • Percent identity refers to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR, Madison WI) which creates alignments between two or more sequences according to methods selected by the user, e.g., the clustal method. (See, e.g., Higgins, D.G. and P.M. Sharp (1988) Gene 73:237-244.) Parameters for each method may be the default parameters provided by MEGALIGN or may be specified by the user. The clustal algorithm groups sequences into clusters by examining the distances between all pairs.
  • the clusters are aligned pairwise and then in groups.
  • the percentage similarity between two amino acid sequences e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity.
  • Percent identity between nucleic acid sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions.
  • HACs Human artificial chromosomes
  • HACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for stable mitotic chromosome segregation and maintenance.
  • humanized antibody refers to antibody molecules in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
  • Hybridization refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
  • Immuno response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
  • microarray refers to an arrangement of distinct polynucleotides on a substrate.
  • modulate refers to a change in the activity of TRNSFS. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of TRNSFS.
  • nucleic acid refers to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
  • fragment refers to those nucleic acid sequences which comprise a region of unique polynucleotide sequence that specifically identifies SEQ ID NO: 16-30, for example, as distinct from any other sequence in the same genome.
  • oligonucleotide refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray.
  • Oligonucleotide is substantially equivalent to the terms “amplimer,” “primer,” “oligomer,” and “probe,” as these terms are commonly defined in the art.
  • sample is used in its broadest sense.
  • a sample suspected of containing nucleic acids encoding TRNSFS, or fragments thereof, or TRNSFS itself, may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
  • Stringent conditions can be defined by salt concentration, the concentration of organic solvent, e.g., formamide, temperature, and other conditions well known in the art.
  • stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature.
  • substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably about 75% free, and most preferably about 90% free from other components with which they are naturally associated.
  • substitution refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
  • Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
  • the substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • Transformation describes a process by which exogenous DNA enters and changes a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell.
  • the method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, viral infection, electroporation, heat shock, lipofection, and particle bombardment.
  • the term "transformed” cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
  • TRNSFS polypeptides refers to an amino acid sequence that is altered by one or more amino acid residues.
  • the variant may have "conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "nonconservative” changes (e.g., replacement of glycine with tryptophan).
  • Analogous minor variations may also include amino acid deletions or insertions, or both.
  • Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).
  • variants when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to TRNSFS. This definition may also include, for example, "allelic” (as defined above), “splice,” “species,” or “polymorphic” variants.
  • a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or an absence of domains.
  • Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other.
  • a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
  • Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
  • SNPs single nucleotide polymorphisms
  • the invention is based on the discovery of new human human transferase proteins
  • TRNSFS the polynucleotides encoding TRNSFS
  • Table 1 lists the Incyte clones used to assemble full length nucleotide sequences encoding
  • TRNSFS Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of the polypeptide and nucleotide sequences, respectively.
  • Column 3 shows the clone IDs of the Incyte clones in which nucleic acids encoding each TRNSFS were identified, and column 4 shows the cDNA libraries from which these clones were isolated.
  • Column 5 shows Incyte clones and their corresponding cDNA libraries. Clones for which cDNA libraries are not indicated were derived from pooled cDNA libraries. The clones in column 5 were used to assemble the consensus nucleotide sequence of each TRNSFS and are useful as fragments in hybridization technologies.
  • column 1 references the SEQ ID NO; column 2 shows the number of amino acid residues in each polypeptide; column 3 shows potential phosphorylation sites; column 4 shows potential glycosylation sites; column 5 shows the amino acid residues comprising signature sequences and motifs; column 6 shows homologous sequences as identified by BLAST analysis; and column 7 shows analytical methods used to characterize each polypeptide through sequence homology and protein motifs.
  • SEQ ID NO: l has chemical and structural similarity with human myristoyl CoA:protein N-myristoyltransferase (GI 2443814; SEQ ID NO:31).
  • SEQ ID NO: 1 and human myristoyl CoA:protein N- myristoyltransferase share 74% identity, share two potential N-glycosylation sites, three potential casein kinase II phosphorylation sites, seven potential protein kinase C phosphorylation sites, the myristoyl CoA:protein N-myristoyltransferase signatures, and have similar isoelectric points, 7.7 and 8.2, respectively.
  • SEQ ID NO:2 also has one potential bacterial hexapeptide-transferase signature from residue V 256 to V 284 This signature is conserved among a number of bacterial transferases which are believed to belong to a single family and are involved in the biosynthesis of glycolipids, polysaccharides, and other macromolecules. As shown in Figures 2A, 2B, 2C and 2D, SEQ ID NO:2 has chemical and structural similarity with Arabidopsis thaliana MPG (GI 2642159; SEQ ID NO:32), and Caenorhabditis elegans MPG (GI 2804432; SEQ ID NO:33).
  • SEQ ID NO:2 and Arabidopsis thaliana MPG share 61% identity
  • MPGh and Caenorhabditis elegans MPG share 63% identity.
  • the ADP-glucose pyrophosphorylase signature of MPGh is also conserved within both, Arabidopsis thaliana and Caenorhabditis elegans MPGs.
  • the potential N-glycosylation site at residue N 322 , the potential casein kinase II phosphorylation sites at residues S 78 , T 136 and T 19 erase the potential tyrosine kinase phosphorylation site at residue Y 144 , and the potential bacterial hexapeptide-transferase signature of SEQ ID NO:2 are conserved in both Arabidopsis thaliana and Caenorhabditis elegans MPGs.
  • the columns of Table 3 show the tissue-specificity and diseases, disorders, or conditions associated with nucleotide sequences encoding TRNSFS.
  • the first column of Table 3 lists the nucleotide SEQ ID NOs.
  • Column 2 lists fragments of the nucleotide sequences of column 1. These fragments are useful, for example, in hybridization or amplification technologies to identify SEQ ID NO: 16-30 and to distinguish between SEQ ID NO: 16-30 and related polynucleotide sequences.
  • the polypeptides encoded by these fragments are useful, for example, as immunogenic peptides.
  • Column 3 lists tissue categories which express TRNSFS as a fraction of total tissues expressing TRNSFS.
  • Column 4 lists diseases, disorders, or conditions associated with those tissues expressing TRNSFS as a fraction of total tissues expressing TRNSFS.
  • Column 5 lists the vectors used to subclone each cDNA library.
  • SEQ ID NO:l in reproductive, smooth muscle, and nervous tissue.
  • SEQ ID NO:2 in reproductive and gastrointestinal tissue.
  • Table 4 show descriptions of the tissues used to construct the cDNA libraries from which cDNA clones encoding TRNSFS were isolated.
  • Column 1 references the nucleotide SEQ ID NOs
  • column 2 shows the cDNA libraries from which these clones were isolated
  • column 3 shows the tissue origins and other descriptive information relevant to the cDNA libraries in column 2.
  • TRNSFS variants are one which has at least about 80%, more preferably at least about 90%, and most preferably at least about 95% amino acid sequence identity to the TRNSFS amino acid sequence, and which contains at least one functional or structural characteristic of TRNSFS.
  • a variety of expression vector/host systems may be utilized to contain and express sequences encoding TRNSFS. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus,TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
  • the invention is not limited by the host cell employed.
  • a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding TRNSFS.
  • routine cloning, subcloning, and propagation of polynucleotide sequences encoding TRNSFS can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding TRNSFS into the vector's multiple cloning site disrupts the lacL gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules.
  • these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
  • vectors which direct high level expression of TRNSFS may be used.
  • vectors containing the strong, inducible T5 or T7 bacteriophage promoter may be used.
  • Yeast expression systems may be used for production of TRNSFS.
  • a number of vectors containing constitutive or inducible promoters may be used in the yeast Saccharomvces cerevisiae or Pichia pastoris.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters
  • such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
  • Plant systems may also be used for expression of TRNSFS. Transcription of sequences encoding TRNSFS may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Brogue, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ.
  • viral promoters e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:1311).
  • plant promoters such
  • constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (See, e.g.. The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196.)
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer
  • RSV Rous sarcoma virus
  • SV40 or EBV-based vectors may also be used for high-level protein expression.
  • Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al. (1997) Nat. Genet.
  • Host cells transformed with nucleotide sequences encoding TRNSFS may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode TRNSFS may be designed to contain signal sequences which direct secretion of TRNSFS through a prokaryotic or eukaryotic cell membrane.
  • a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro" form of the protein may also be used to specify protein targeting, folding, and/or activity.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas, VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • nucleic acid sequences encoding TRNSFS may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric TRNSFS protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of TRNSFS activity.
  • Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices.
  • Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
  • a fusion protein may also be engineered to contain a proteolytic cleavage site located between the TRNSFS encoding sequence and the heterologous protein sequence, so that TRNSFS may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995. supra, ch 10). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
  • TRNSFS may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton. supra, pp. 55-60.) Protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be achieved, for example, using the ABI 431 A peptide synthesizer (Perkin-Elmer). Various fragments of TRNSFS may be synthesized separately and then combined to produce the full length molecule. THERAPEUTICS Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of TRNSFS and human transferase proteins.
  • TRNSFS or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of TRNSFS.
  • disorders include, but are not limited to, a cancer, such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; a developmental disorder, such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis
  • an agonist which modulates the activity of TRNSFS may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of TRNSFS including, but not limited to, those listed above.
  • an antagonist of TRNSFS may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of TRNSFS. Examples of such disorders include, but are not limited to, those described above.
  • an antibody which specifically binds TRNSFS may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express TRNSFS.
  • a vector expressing the complement of the polynucleotide encoding TRNSFS may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of TRNSFS including, but not limited to, those described above.
  • any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • TRNSFS An antagonist of TRNSFS may be produced using methods which are generally known in the art.
  • purified TRNSFS may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind TRNSFS.
  • Antibodies to TRNSFS may also be generated using methods that are well known in the art.
  • Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library.
  • Neutralizing antibodies i.e., those which inhibit dimer formation are especially preferred for therapeutic use.
  • the oligopeptides, peptides, or fragments used to induce antibodies to TRNSFS have an amino acid sequence consisting of at least about 5 amino acids, and, more preferably, of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of TRNSFS amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced. Monoclonal antibodies to TRNSFS may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture.
  • hybridoma technique examples include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV- hybridoma technique.
  • chimeric antibodies such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity.
  • techniques developed for the production of single chain antibodies may be adapted, using methods known in the art, to produce TRNSFS-specific single chain antibodies.
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton D.R. (1991) Proc. Natl. Acad. Sci. 88:10134-10137.) Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G. et al.
  • immunoassays may be used for screening to identify antibodies having the desired specificity.
  • Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.
  • Such immunoassays typically involve the measurement of complex formation between TRNSFS and its specific antibody.
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering TRNSFS epitopes is preferred, but a competitive binding assay may also be employed (Pound, supra).
  • K a is defined as the molar concentration of TRNSFS-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions.
  • K a association constant
  • the K a determined for a preparation of monoclonal antibodies, which are monospecific for a particular TRNSFS epitope represents a true measure of affinity.
  • High-affinity antibody preparations with K a ranging from about 10 9 to 10 12 L/mole are preferred for use in immunoassays in which the TRNSFS-antibody complex must withstand rigorous manipulations.
  • Low-affinity antibody preparations with K a ranging from about 10 6 to 10 7 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of TRNSFS, preferably in active form, from the antibody (Catty, D. (1988) Antibodies. Volume I: A Practical Approach. IRL Press, Washington, DC; Liddell, J. E. and Cryer, A. (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
  • polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications.
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is preferred for use in procedures requiring precipitation of TRNSFS-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available. (See, e.g., Catty, supra, and Coligan et al. supra.)
  • the polynucleotides encoding TRNSFS may be used for therapeutic purposes.
  • the complement of the polynucleotide encoding TRNSFS may be used in situations in which it would be desirable to block the transcription of the mRNA.
  • cells may be transformed with sequences complementary to polynucleotides encoding TRNSFS.
  • complementary molecules or fragments may be used to modulate TRNSFS activity, or to achieve regulation of gene function.
  • sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding TRNSFS.
  • Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector, and may last even longer if appropriate replication elements are part of the vector system.
  • a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding TRNSFS.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • RNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding TRNSFS. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutive ly or inducibly, can be introduced into cell lines, cells, or tissues.
  • RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al. ( 1997) Nature Biotechnology 15 :462-466.)
  • any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • compositions may consist of TRNSFS, antibodies to TRNSFS, and mimetics, agonists, antagonists, or inhibitors of TRNSFS.
  • the compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone, or in combination with other agents, drugs, or hormones.
  • compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA).
  • compositions for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores.
  • auxiliaries can be added, if desired.
  • Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate.
  • Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic acids. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • compositions After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition.
  • labeling would include amount, frequency, and method of administration.
  • a therapeutically effective dose refers to that amount of active ingredient, for example TRNSFS or fragments thereof, antibodies of TRNSFS, and agonists, antagonists or inhibitors of TRNSFS, which ameliorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeutically effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD 5 JED 50 ratio.
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration. The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.
  • antibodies which specifically bind TRNSFS may be used for the diagnosis of disorders characterized by expression of TRNSFS, or in assays to monitor patients being treated with TRNSFS or agonists, antagonists, or inhibitors of TRNSFS.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for TRNSFS include methods which utilize the antibody and a label to detect TRNSFS in human body fluids or in extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • TRNSFS A variety of protocols for measuring TRNSFS, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of TRNSFS expression.
  • Normal or standard values for TRNSFS expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to TRNSFS under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, preferably by photometric means. Quantities of TRNSFS expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
  • the polynucleotides encoding TRNSFS may be used for diagnostic purposes.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding TRNSFS or closely related molecules may be used to identify nucleic acid sequences which encode TRNSFS.
  • the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low), will determine whether the probe identifies only naturally occurring sequences encoding TRNSFS, allelic variants, or related sequences.
  • Probes may also be used for the detection of related sequences, and should preferably have at least 50% sequence identity to any of the TRNSFS encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO: 16-30 or from genomic sequences including promoters, enhancers, and introns of the TRNSFS gene.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
  • oligonucleotides designed from the sequences encoding TRNSFS may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding TRNSFS, or a fragment of a polynucleotide complementary to the polynucleotide encoding TRNSFS, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantitation of closely related DNA or RNA sequences.
  • oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as targets in a microarray.
  • the microarray can be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.
  • nucleic acid sequences encoding TRNSFS may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence.
  • the sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDNA libraries.
  • Fluorescent in situ hybridization may be correlated with other physical chromosome mapping techniques and genetic map data.
  • FISH Fluorescent in situ hybridization
  • Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) site. Correlation between the location of the gene encoding TRNSFS on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder.
  • the nucleotide sequences of the invention may be used to detect differences in gene sequences among normal, carrier, and affected individuals.
  • In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques.
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
  • TRNSFS in another embodiment, TRNSFS, its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques.
  • the fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly.
  • the formation of binding complexes between TRNSFS and the agent being tested may be measured.
  • Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT application WO84/03564.) In this method, large numbers of different small test compounds are synthesized on a solid substrate.
  • test compounds are reacted with TRNSFS, or fragments thereof, and washed. Bound TRNSFS is then detected by methods well known in the art. Purified TRNSFS can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • nucleotide sequences which encode TRNSFS may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
  • poly(A+) RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
  • RNA was provided with RNA and constructed the corresponding cDNA libraries.
  • cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
  • cDNA was size-selected (300-1000 bp) using SEPHACRYL SI 000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis.
  • cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), pSPORTl plasmid (Life Technologies), or pINCY (Incyte Pharmaceuticals, Palo Alto CA).
  • Recombinant plasmids were transformed into competent E. coli cells including XL 1 -Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Life Technologies. II. Isolation of cDNA Clones
  • Plasmids were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8, QIAWELL 8 Plus, QIAWELL 8 Ultra plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C
  • plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384- well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland). III.
  • cDNA sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Perkin-Elmer) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system.
  • cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
  • Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing systems (Perkin-Elmer) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997. supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example V. The polynucleotide sequences derived from cDNA sequencing were assembled and analyzed using a combination of software programs which utilize algorithms well known to those skilled in the art.
  • the polynucleotide sequences were validated by removing vector, linker, and polyA sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programing, and dinucleotide nearest neighbor analysis. The sequences were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS to acquire annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences were assembled into full length polynucleotide sequences using programs based on Phred, Phrap, and Consed, and were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA.
  • HMM Hidden Markov Model
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound.
  • a membrane on which RNAs from a particular cell type or tissue have been bound See, e.g., Sambrook. supra, ch. 7; Ausubel, 1995, supra, ch. 4 and 16.
  • Analogous computer techniques applying BLAST were used to search for identical or related molecules in nucleotide databases such as GenBank or LIFESEQ (Incyte Pharmaceuticals). This analysis is much faster than multiple membrane-based hybridizations.
  • the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar.
  • the basis of the search is the product score, which is defined as: % sequence identity x % maximum BLAST score
  • the product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1% to 2% error, and, with a product score of 70, the match will be exact. Similar molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.
  • the results of northern analyses are reported as a percentage distribution of libraries in which the transcript encoding TRNSFS occurred.
  • Analysis involved the categorization of cDNA libraries by organ/tissue and disease.
  • the organ/tissue categories included cardiovascular, dermatologic, developmental, endocrine, gastrointestinal, hematopoietic/immune, musculoskeletal, nervous, reproductive, and urologic.
  • the disease/condition categories included cancer, inflammation/trauma, cell proliferation, fetal, neurological, and pooled. For each category, the number of libraries expressing the sequence of interest was counted and divided by the total 5 number of libraries across all categories. Percentage values of tissue-specific and disease- or condition-specific expression are reported in Table 3. V. Extension of TRNSFS Encoding Polynucleotides
  • oligonucleotide primers designed from this fragment.
  • One primer was synthesized to initiate 5' extension of the known fragment, and the other primer, to initiate 3' extension of the known fragment.
  • the initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about
  • the plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
  • a 5 ⁇ l to 10 ⁇ l aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose mini-gel to determine which reactions were successful in extending the sequence.
  • the extended nucleotides were desalted and concentrated, transferred to 384- well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech).
  • CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
  • sonicated or sheared prior to religation into pUC 18 vector
  • the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega).
  • Extended clones were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, individual colonies were picked and cultured overnight at 37°C in 384-well plates in LB/2x carb liquid media.
  • the cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1 : 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 72°C, 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the same conditions as described above.
  • nucleotide sequences of SEQ ID NO: 16 and of SEQ ID NO: 18-30 are used to obtain 5' regulatory sequences using the procedure above, oligonucleotides designed for such extension, and an appropriate genomic library.
  • the full length nucleic acid sequence of SEQ ID NO: 17 was produced by extension of an appropriate fragment of the full length molecule, using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate extension of an antisense polynucleotide, and the other was synthesized to initiate extension of a sense polynucleotide. Primers were used to facilitate the extension of the known sequence "outward" generating amplicons containing new unknown nucleotide sequence for the region of interest.
  • the initial primers were designed from the cDNA using OLIGO 4.06 software (National Biosciences, Madison, MN), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
  • Step 3 68° C for 6 min
  • Step 4 94° C for 15 sec
  • Step 10 68° C for 7: 15 min Step 1 1 Repeat steps 8 through 10 for an additional 12 cycles
  • PCR amplification 18 ⁇ l of concentrated PCR reaction mix (3.3x) containing 4 units of rTth DNA polymerase, a vector primer, and one or both of the gene specific primers used for the extension reaction were added to each well. Amplification was performed using the following conditions: Step 1 94° C for 60 sec
  • Step 2 94° C for 20 sec
  • Step 5 Repeat steps 2 through 4 for an additional 29 cycles Step 6 72° C for 180 sec
  • nucleotide sequence of SEQ ID NO: 17 is used to obtain 5' regulatory sequences using the procedure above, oligonucleotides designed for 5' extension, and an appropriate genomic library.
  • Hybridization probes derived from SEQ ID NO: 16-30 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments.
  • Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech).
  • the DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40 °C. To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized using autoradiography and compared.
  • a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
  • a complementary oligonucleotide is designed to prevent ribosomal binding to the TRNSFS-encoding transcript.
  • EX. Expression of TRNSFS Expression and purification of TRNSFS is achieved using bacterial or virus-based expression systems. For expression of TRNSFS in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
  • promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
  • Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
  • Antibiotic resistant bacteria express TRNSFS upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG).
  • IPTG isopropyl beta-D-thiogalactopyranoside
  • Expression of TRNSFS in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus.
  • AcMNPV Autographica californica nuclear polyhedrosis virus
  • FLAG an 8-amino acid peptide
  • 6-His a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel (1995. supra, ch 10 and 16). Purified TRNSFS obtained by these methods can be used directly in the following activity assay. X. Demonstration of TRNSFS Activity
  • the mixture is incubated for two hours at 37°C
  • the reaction products are adjusted to 0.25 M ammonium formate, pH 4.0, and separated on a C18 reverse phase chromatography column.
  • the column is washed and the products are eluted with 70% methanol.
  • the radioactivity recovered in the acceptor molecule is measured using a liquid scintillation counter and is proportional to the activity of HNK sulfotransferase in the assay.
  • CDP-ethanolamine and diphosphate are measured with chromatographic methods, whereby the reaction products are separated from the substrates.
  • the amounts of CDP-ethanolamine and diphosphate produced are directly proportional to the activity of TRNSFS 5 in biological samples.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
  • FCM Flow cytometry
  • an automated, laser optics-based technique is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death.
  • TRNSFS The influence of TRNSFS on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding TRNSFS and either CD64 or CD64- GFP.
  • CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG).
  • Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
  • mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding TRNSFS and other genes of interest can be analyzed by Northern analysis or microarray techniques. XII. Production of TRNSFS Specific Antibodies
  • TRNSFS amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art.
  • LASERGENE software DNASTAR
  • Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel, 1995. supra, ch. 11.)
  • oligopeptides 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to KLH (Sigma-Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity.
  • ABI 431 A peptide synthesizer Perkin-Elmer
  • KLH Sigma-Aldrich, St. Louis MO
  • MBS N-maleimidobenzoyl-N-hydroxysuccinimide ester
  • Rabbits are immunized with the oligopeptide- KLH complex in complete Freund's adjuvant.
  • Resulting antisera are tested for antipeptide activity by, for example, binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
  • XIII Purification of Naturally Occurring TRNSFS Using Specific Antibodies
  • Naturally occurring or recombinant TRNSFS is substantially purified by immunoaffinity chromatography using antibodies specific for TRNSFS.
  • An immunoaffinity column is constructed by covalently coupling anti-TRNSFS antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
  • TRNSFS or biologically active fragments thereof, are labeled with 125 I Bolton-Hunter reagent.
  • Bolton-Hunter reagent See, e.g., Bolton et al. (1973) Biochem. J. 133:529.
  • Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled TRNSFS, washed, and any wells with labeled TRNSFS complex are assayed. Data obtained using different concentrations of TRNSFS are used to calculate values for the number, affinity, and association of TRNSFS with the candidate molecules.
  • ABI FACTURA A program that removes vector sequences and masks Perkin-Elmer Applied Biosystems, ambiguous bases in nucleic acid sequences. Foster City, CA.
  • ABI/PARACEL A Fast Data Finder useful in comparing and annotating Perkin-Elmer Applied Biosystems, Mismatch ⁇ 50%
  • ABI AutoAssembler A program that assembles nucleic acid sequences. Perkin-Elmer Applied Biosystems, Foster City, CA.
  • Phred A base-calling algorithm that examines automated Ewing, B. et al. (1998) Genome sequencer traces with high sensitivity and probability. Res. 8:175-185; Ewing, B. and P. Green (1998) Genome Res. 8: 186- 194.

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Abstract

La présente invention concerne des transférases humaines (TRNFS) et des polynucléotides qui identifient et codent les TRNFS. L'invention concerne également des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes et des antagonistes. L'invention concerne enfin le diagnostic, le traitement et la prévention de troubles liés à l'expression des TRNFS.
PCT/US1999/020989 1998-09-10 1999-09-09 Transferases humaines WO2000014251A2 (fr)

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EP99948200A EP1109917A2 (fr) 1998-09-10 1999-09-09 Transferases humaines
JP2000568992A JP2004512804A (ja) 1998-09-10 1999-09-09 ヒト転移酵素タンパク質
US09/786,240 US6558935B1 (en) 1998-09-10 1999-09-09 Human transferase proteins
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WO2001049862A1 (fr) * 1999-12-29 2001-07-12 Fudan University Nouveau polypeptide, famille proteique 10 d'hormones de croissance, et polynucleotide codant pour ce polypeptide
WO2001066759A2 (fr) * 2000-03-07 2001-09-13 Millennium Pharmaceuticals, Inc. 26886, nouvel element de la famille de la carnitine acyltransferase et ses utilisations
WO2002020801A2 (fr) * 2000-09-01 2002-03-14 Millennium Pharmaceuticals Inc. 26199, 33530, 33949, 47148, 50226, et 58764, nouveaux membres de la famille des transferases humaines et utilisations de ceux-ci
WO2002031159A1 (fr) * 2000-10-06 2002-04-18 Kirin Beer Kabushiki Kaisha Nouvelle n-acetylglucosaminyltransferase et polynucleotide codant pour celle-ci
WO2002081633A2 (fr) * 2001-04-04 2002-10-17 University Of Iowa Research Foundation Gene de susceptibilite au syndrome de bardet-biedl et utilisations associees
WO2004071411A3 (fr) * 2003-02-04 2005-03-03 Millennium Pharm Inc Procede et compositions pour traiter la douleur et les troubles douloureux en utilisant 16386, 15402, 21165, 1423, 636, 12303, 21425, 27410, 38554, 38555, 55063, 57145, 59914, 94921, 16852, 33260, 58573, 30911, 85913, 14303, 16816, 17827 or 32620
WO2005030947A3 (fr) * 2003-09-30 2005-06-23 Evotec Neurosciences Gmbh Utilisation diagnostique et therapeutique d'une sulfotransferase pour des maladies neurodegeneratives
US6933142B1 (en) * 1998-03-20 2005-08-23 The Regents Of The University Of California Hec-g1cnac6st
US6967093B2 (en) 1998-03-20 2005-11-22 The Regents Of The University Of California Glycosyl sulfotransferase-3
WO2008097585A2 (fr) * 2007-02-06 2008-08-14 Dalhousie University Mutations dans le gène humain ubiad1
US7432345B2 (en) 1998-11-17 2008-10-07 Genentech, Inc. PRO1475 polypeptide
US7736654B2 (en) 2001-04-10 2010-06-15 Agensys, Inc. Nucleic acids and corresponding proteins useful in the detection and treatment of various cancers
US9187552B2 (en) 2010-05-27 2015-11-17 Merck Sharp & Dohme Corp. Method for preparing antibodies having improved properties
US9328170B2 (en) 2011-05-25 2016-05-03 Merck Sharp & Dohme Corp. Method for preparing Fc containing polypeptides having improved properties

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US6933142B1 (en) * 1998-03-20 2005-08-23 The Regents Of The University Of California Hec-g1cnac6st
US6967093B2 (en) 1998-03-20 2005-11-22 The Regents Of The University Of California Glycosyl sulfotransferase-3
US7432345B2 (en) 1998-11-17 2008-10-07 Genentech, Inc. PRO1475 polypeptide
WO2001049862A1 (fr) * 1999-12-29 2001-07-12 Fudan University Nouveau polypeptide, famille proteique 10 d'hormones de croissance, et polynucleotide codant pour ce polypeptide
WO2001066759A2 (fr) * 2000-03-07 2001-09-13 Millennium Pharmaceuticals, Inc. 26886, nouvel element de la famille de la carnitine acyltransferase et ses utilisations
WO2001066759A3 (fr) * 2000-03-07 2002-05-02 Millennium Pharm Inc 26886, nouvel element de la famille de la carnitine acyltransferase et ses utilisations
WO2002020801A2 (fr) * 2000-09-01 2002-03-14 Millennium Pharmaceuticals Inc. 26199, 33530, 33949, 47148, 50226, et 58764, nouveaux membres de la famille des transferases humaines et utilisations de ceux-ci
WO2002020801A3 (fr) * 2000-09-01 2003-06-19 Millennium Pharm Inc 26199, 33530, 33949, 47148, 50226, et 58764, nouveaux membres de la famille des transferases humaines et utilisations de ceux-ci
WO2002031159A1 (fr) * 2000-10-06 2002-04-18 Kirin Beer Kabushiki Kaisha Nouvelle n-acetylglucosaminyltransferase et polynucleotide codant pour celle-ci
US7217548B2 (en) 2000-10-06 2007-05-15 National Institute Of Advanced Industrial Science And Technology N-acetylglucosaminlytransferase and polynucleotide encoding the same
US7045317B2 (en) 2001-04-04 2006-05-16 University Of Iowa Research Foundation Bardet-biedl susceptibility gene and uses thereof
WO2002081633A2 (fr) * 2001-04-04 2002-10-17 University Of Iowa Research Foundation Gene de susceptibilite au syndrome de bardet-biedl et utilisations associees
WO2002081633A3 (fr) * 2001-04-04 2003-07-17 Univ Iowa Res Found Gene de susceptibilite au syndrome de bardet-biedl et utilisations associees
US7736654B2 (en) 2001-04-10 2010-06-15 Agensys, Inc. Nucleic acids and corresponding proteins useful in the detection and treatment of various cancers
WO2004071411A3 (fr) * 2003-02-04 2005-03-03 Millennium Pharm Inc Procede et compositions pour traiter la douleur et les troubles douloureux en utilisant 16386, 15402, 21165, 1423, 636, 12303, 21425, 27410, 38554, 38555, 55063, 57145, 59914, 94921, 16852, 33260, 58573, 30911, 85913, 14303, 16816, 17827 or 32620
US7169751B2 (en) 2003-02-04 2007-01-30 Bayer Healthcare Ag Method of identifying a compound capable of treating a pain or a painful disorder
JP2007508810A (ja) * 2003-09-30 2007-04-12 エヴォテック ニューロサイエンシス ゲゼルシャフト ミット ベシュレンクテル ハフツング スルホトランスフェラーゼの神経変性疾患に関する診断的および治療的使用
WO2005030947A3 (fr) * 2003-09-30 2005-06-23 Evotec Neurosciences Gmbh Utilisation diagnostique et therapeutique d'une sulfotransferase pour des maladies neurodegeneratives
WO2008097585A2 (fr) * 2007-02-06 2008-08-14 Dalhousie University Mutations dans le gène humain ubiad1
WO2008097585A3 (fr) * 2007-02-06 2008-12-31 Univ Dalhousie Mutations dans le gène humain ubiad1
US8334369B2 (en) 2007-02-06 2012-12-18 Genome Atlantic Mutations in human UBIAD1
US9187552B2 (en) 2010-05-27 2015-11-17 Merck Sharp & Dohme Corp. Method for preparing antibodies having improved properties
US10858686B2 (en) 2010-05-27 2020-12-08 Merck Sharp & Dohme Corp. Method for preparing antibodies having improved properties
US11959118B2 (en) 2010-05-27 2024-04-16 Merck Sharp & Dohme Llc Fc-containing polypeptides having improved properties and comprising mutations at positions 243 and 264 of the Fc-region
US9328170B2 (en) 2011-05-25 2016-05-03 Merck Sharp & Dohme Corp. Method for preparing Fc containing polypeptides having improved properties

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