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Definitions

• This invention relates to nucleic acid and ammo acid sequences of synthetases and to the use of these sequences in the diagnosis, treatment, and prevention of immune, neuronal, and reproductive disorders, and cell pro ferative disorders including cancer
• a large number of cellular biosynthetic intermediary metabolism processes involve lntermolecular transfer of carbon atom-containing substrates (carbon substrates) Examples of such reactions include the tncarboxyhc acid cycle, synthesis of fatty acids and long-chain phospholipids, synthesis of alcohols and aldehydes, synthesis of intermediary metabolites, and reactions involved in the amino acid degradation pathways Many of these reactions are catalyzed by synthetases (also called hgases), which catalyze the formation of a bond between two substrate molecules Some of these reactions require input of energy, usually in the form of conversion of ATP to either ADP or AMP and pyrophosphate Synthetases are named for the products of the reaction they catalyze and are involved in such processes as metabolism and the synthesis of macromolecules Ligases forming carbon-oxygen bonds
• Proteins make up more than half of the total dry mass of a cell
• the synthesis of proteins is central to cell maintenance, growth, and development Synthesis occurs on ⁇ bosomes and depends on the cooperative interaction of several classes of RNA molecules
• the process begins with transcription of the genetic code contained within the DNA to form messenger RNA (mRNA)
• mRNA messenger RNA
• the mRNA moves in steps through a ⁇ bosome and the nucleotide sequence of the mRNA is translated into a corresponding sequence of amino acids to construct a distinct protein chain
• the am oacyl-transfer RNA (tRNA) synthetases are important RNA-associated enzymes with roles in translation Protein biosynthesis depends on each amino acid forming a linkage with the appropnate tRNA
• the aminoacyl-tRNA synthetases are responsible for the activation and correct attachment of an amino acid with its cognate tRNA
• the 20 aminoacyl-tRNA synthetase enzymes can be divided into two structural classes, and each class is charactenzed by a distinctive topology of the catalytic domain Class I enzymes contain a catalytic domain based on the nucleotide-binding Rossman 'fold' Class II enzymes contain a central catalytic domain, which consists of a seven-stranded antiparallel ⁇ -sheet motif, as well as N- and C- terminal regulatory domains Class II enzymes are separated into two groups based on the heterodimenc or homodimenc structure of the enzyme, the latter group is further subdivided by the structure
• a carbon substrate is denved from a small molecule containing at least two carbon atoms
• the carbon substrate is often covalently bound to a larger molecule which acts as a carbon substrate earner molecule within the cell
• the earner molecule is coenzyme A
• Coenzyme A (CoA) is structurally related to denvatives of the nucleotide ADP and consists of 4'- ⁇ hosphopantethe ⁇ ne linked via a phosphodiester bond to the alpha phosphate group of adenosine 3',5'-b ⁇ sphosphate
• the terminal thiol group of 4'-phosphopantetheme acts as the site for carbon substrate bond formation
• the predominant carbon substrates which utilize CoA as a earner molecule dunng biosynthesis and intermediary metabolism in the cell are acetyl, succinyl, and propionyl moieties, collectively referred to as acyl groups
• Other carbon substrates include enoyl lipid
• acyl-CoA synthetase activity 0 acetyl-CoA synthetase, which activates acetate and several other low molecular weight carboxylic acids and is found in muscle mitochondna and the cytosol of other tissues, n) medium-chain acyl-CoA synthetase, which activates fatty acids containing between four and eleven carbon atoms (predominantly from dietary sources), and is present only in liver mitochondna, and in) acyl CoA synthetase, which is specific for long chain fatty acids with between six and twenty carbon atoms, and is found m microsomes and the mitochondna Proteins associated with acyl-CoA synthetase activity have been identified from many sources including bactena, yeast, plants, mouse, and man The activity of acyl-CoA synthetase may be modulated by phosphorylation of
• a key representative of the amide synthases is the enzyme glutamine synthetase (glutamate- ammonia hgase) that catalyzes the animation of glutamic acid to glutamme by ammonia using the energy of ATP hydrolysis
• glutamine is the primary source for the amino group in va ⁇ ous amide transfer reactions involved in de novo pyrimidine nucleotide synthesis and in pu ⁇ ne and py ⁇ midine ⁇ bonucleotide interconversions, as well as the conversion of aspartate to asparagine
• Overexpression of glutamine synthetase has been observed in p ⁇ mary liver cancer (Chnsta, L et al ( 1994) Gastroent 106 1312- 1320) Cyclo-hgases and other carbon-nitrogen ligases compnse va ⁇ ous enzymes and enzyme complexes that participate in the de novo pathways to pu ⁇ ne and py ⁇ midine biosynthesis Because these pathways are c ⁇ tical to the synthesis of nu
• de novo synthesis of the py ⁇ midine nucleotides u ⁇ dylate and cytidylate also a ⁇ ses from a common precursor, in this instance the nucleotide orotidylate de ⁇ ved from orotate and phospho ⁇ bosyl pyrophosphate (PPRP)
• PPRP phospho ⁇ bosyl pyrophosphate
• ATCase aspartate transcarbamylase
• carbamyl phosphate synthetase II carbamyl phosphate synthetase II
• DHOase dihydroorotase
• Ligases in this group are represented by the carboxylases acetyl-CoA carboxylase and pyruvate carboxylase
• Acetyl-CoA carboxylase is a complex which includes a biotin carboxyl earner protein, biotm carboxylase, and a carboxyl transferase made up of two alpha and two beta subunits. This complex catalyzes the carboxylation of Acetyl-CoA from CO, and t O using the energy of ATP hydrolysis (PRINTS document PRO 1069).
• Acetyl-CoA carboxylase is the rate-limiting step in the biogenesis of long-chain fatty acids
• Two isoforms of Acetyl-CoA carboxylase, types I and types II, are expressed in humans in a tissue-specific manner (Ha, J. et al. (1994) Eur. J. Biochem. 219:297- 306).
• Pyruvate carboxylase is a nuclear-encoded mitochond ⁇ al enzyme that catalyzes the conversion of pyruvate to oxaloacetate, a key intermediate in the cit ⁇ c acid cycle. Ligases forming phospho ⁇ c ester bonds
• Ligases in this group are represented by the DNA ligases involved in both DNA replication and repair
• DNA ligases seal phosphodiester bonds between two adjacent nucleotides in a DNA chain using the energy from ATP hydrolysis to first activate the free 5'-phosphate of one nucleotide and then react it with the 3'-OH group of the adjacent nucleotide.
• This reseahng reaction is used in both DNA replication to join small DNA fragments called "Okazaki" fragments that are transiently formed in the process of replicating new DNA, and in DNA repair.
• DNA repair is the process by which accidental base changes, such as those produced by oxidative damage, hydrolytic attack, or uncontrolled methylation of DNA, are corrected before replication or transc ⁇ ption of the DNA can occur.
• Bloom's syndrome is an inhe ⁇ ted human disease in which individuals are partially deficient in DNA hgation and consequently have an increased incidence of cancer (Alberts, B et al. ( 1994) The Molecular Biology of the Cell, Garland Publishing Inc., New York, NY, p. 247)
• the discovery of new synthetases and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful m the diagnosis, prevention, and treatment of immune, neuronal, and reproductive disorders, and cell prohferative disorders including cancer.
• the invention features punfied polypeptides, human synthetases, referred to collectively as
• the invention provides an isolated polypepttde compnsing an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, b) a naturally occumng amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO.1-15, and d) an immunogenic fragment of an ammo acid sequence selected from the group consisting of SEQ ID NO: 1-15.
• the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1-15.
• the invention further provides an isolated polynucleotide encoding a polypeptide compnsing an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, b) a naturally occumng amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, and d) an immunogenic fragment of an am o acid sequence selected from the group consisting of SEQ ID NO: 1-15.
• the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO- 1-15
• the polynucleotide is selected from the group consisting of SEQ ID NOT 6-30.
• the invention provides a recombinant polynucleotide compnsing a promoter sequence operably linked to a polynucleotide encoding a polypeptide compnsing an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: l-15, b) a naturally occumng amino acid sequence having at least 90% sequence identity to an ammo acid sequence selected from the group consisting of SEQ ID NO 1-15, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1- 15, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-15.
• the invention provides a cell transformed with the recombinant polynucleotide.
• the invention provides a transgenic organism compnsing the recombinant polynucleotide
• the invention also provides a method for producing a polypeptide compnsing an am o acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOT-15, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO- 1-15, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15.
• the method compnses a) cultu ⁇ ng a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide compnsing a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recove ⁇ ng the polypeptide so expressed.
• the invention provides an isolated antibody which specifically binds to a polypeptide compnsing an amino acid sequence selected from the group consisting of a) an ammo acid sequence selected from the group consisting of SEQ ID NO: 1-15, b) a naturally occumng ammo acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO 1-15, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-15, and d) an immunogenic fragment of an ammo acid sequence selected from the group consisting of SEQ ID NO 1-15
• the invention further provides an isolated polynucleotide compnsing a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO 16-30, b) a naturally occu ng polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the
• the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO 16-30, b) a naturally occumng polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO 16-30, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d)
• the method comprises a) hybndizing the sample with a probe compnsing at least 20 contiguous nucleotides compnsing a sequence complementary to said target polynucleotide in the sample, and which probe specifically
• the invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide compnsing an amino acid sequence selected from the group consisting of a) an ammo acid sequence selected from the group consisting of SEQ ID NO 1-15, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO 1-15, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-15, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-15
• the method compnses a) exposing a sample compnsing the polypeptide to a compound, and b) detecting agonist activity in the sample
• the invention provides a pharmaceutical composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient
• the invention provides a method of treating a disease or condition associated with decreased expression of functional SYNT, compnsing admimstenng
• the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide compnsing an ammo acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO 1-15, b) a naturally occumng ammo acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO 1-15, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-15, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-15
• the method compnses a) exposing a sample compnsing the polypeptide to a compound, and b) detecting antagonist activity in the sample
• the invention provides a pharmaceutical composition compnsing an antagonist compound identified by the method and a pharmaceutically acceptable excipient
• the invention provides a method of treating a disease or condition associated with overexpression of functional SYNT, compnsing
• the invention further provides a method of screening for a compound that modulates the activity of a polypeptide comprising an ammo acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO 1- 15, b) a naturally occumng amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO 1-15, c) a biologically active fragment of an ammo acid sequence selected from the group consisting of SEQ ID NO 1- 15, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1- 15
• the method compnses a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) companng the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of
• the invention further provides a method for screening a compound for effectiveness in alternativeng expression of a target polynucleotide, wherein said target polynucleotide compnses a sequence selected from the group consisting of SEQ ID NO 16-30, the method compnsing a) exposing a sample compnsing the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide
• the invention further provides a method for assessing toxicity of a test compound, said method compnsing a) treating a biological sample containing nucleic acids with the test compound, b) hybndizing the nucleic acids of the treated biological sample with a probe compnsing at least 20 contiguous nucleotides of a polynucleotide compnsing a polynucleotide sequence selected from the group consisting of I) a polynucleotide sequence selected from the group consisting of SEQ ID NO 16-30, n) a naturally occumng polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO 16-30, in) a polynucleotide sequence complementary to I), iv) a polynucleotide sequence complementary to n), and v) an RNA equivalent of ⁇ )- ⁇ v) Hybndization occurs under conditions whereby a specific hyb
• Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ ID NOs), clone identification numbers (clone IDs), cDNA hbranes, and cDNA fragments used to assemble full- length sequences encoding SYNT
• Table 2 shows features of each polypeptide sequence, including potential motifs, homologous sequences, and methods, algorithms, and searchable databases used for analysis of SYNT
• Table 3 shows selected 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 hbranes from which cDNA clones encoding SYNT were isolated
• Table 5 shows the tools, programs, and algonthms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters
• SYNT refers to the ammo acid sequences of substantially pu ⁇ fied SYNT obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, munne, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant
• agonist refers to a molecule which intensifies or mimics the biological activity of SYNT Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of SYNT either by directly interacting with SYNT or by acting on components of the biological pathway in which SYNT participates
• Allehc va ⁇ ant is an alternative form of the gene encoding SYNT Allehc va ⁇ ants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered
• a gene may have none, one, or many allehc vanants of its naturally occumng form
• Common mutational changes which give nse to allehc va ⁇ ants are generally ascribed to natural deletions, additions, or substitutions of nucleotides Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence
• altered nucleic acid sequences encoding SYNT include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as SYNT or a polypeptide with at least one functional characte ⁇ stic of SYNT Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding SYNT, and improper or unexpected hybndization to allehc vanants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding SYNT
• the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent SYNT Deliberate amino acid substitutions may be made on the basis of simila ⁇ ty in polarity, charge, solubility, hydrophobicity,
• Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagme and glutamine; and senne and threonine. Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine. and valine; glycine and alanine, and phenylalanine and tyrosine.
• amino acid sequence refers to an ohgopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occumng or synthetic molecules. Where "amino acid sequence” is recited to refer to a sequence of a naturally occumng protein molecule, “ammo 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.
• Amplification relates to the production of additional copies of a nucleic acid sequence Amplification is generally earned out using polymerase chain reaction (PCR) technologies well known in the art.
• PCR polymerase chain reaction
• Antagonist refers to a molecule which inhibits or attenuates the biological activity of SYNT Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of SYNT either by directly interacting with SYNT or by acting on components of the biological pathway in which SYNT participates
• antibody refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
• Antibodies that bind SYNT polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
• the polypeptide or ohgopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
• an animal e.g., a mouse, a rat, or a rabbit
• an animal e.g., a mouse, a rat, or a rabbit
• peptides Commonly used earners that are chemically coupled to peptides include bovine serum albumin, thyroglobu n, and keyhole limpet hemocyanin (KLH).
• KLH keyhole limpet hemocyanin
• the coupled peptide is then used to immunize the animal.
• antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
• an antigenic determinant may compete with the intact antigen (i.e., the lmmunogen used to elicit the immune response) for binding to an antibody
• antisense refers to any composition capable of base-pai ⁇ ng with the "sense" (coding) strand of a specific nucleic acid sequence.
• Antisense compositions may include DNA, RNA; peptide nucleic acid (PNA); ohgonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; o gonucleotides having modified sugar groups such as 2 -methoxyethyl sugars or 2'-methoxyethoxy sugars; or ohgonucleotides having modified bases such as 5-methyl cytosine, 2 -deoxyuracil, or 7-deaza-2 -deoxyguanosine.
• Antisense molecules may be produced by any method including chemical synthesis or transcription Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occumng nucleic acid sequence produced by the cell to form duplexes which block either transc ⁇ ption or translation.
• the designation "negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
• biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occumng molecule.
• immunologically active or “immunogenic” refers to the capability of the natural, recombinant, or synthetic SYNT, or of any ohgopeptide thereof, to induce a specific immune response in appropnate animals or cells and to bind with specific antibodies.
• “Complementary” descnbes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'
• 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 comp ⁇ se a dry formulation or an aqueous solution.
• Compositions compnsing polynucleotide sequences encoding SYNT or fragments of SYNT may be employed as hybndization probes.
• the probes may be stored in freeze-dned form and may be associated with a stabilizing agent such as a carbohydrate
• 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.)
• Consensus sequence refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (PE Biosystems, Foster City CA) in the 5' and or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WA) Some sequences have been both extended and assembled to produce the consensus sequence.
• GELVIEW fragment assembly system GELVIEW fragment assembly system
• Phrap Universality of Washington, Seattle WA
• Constant amino acid substitutions are those substitutions that are predicted to least interfere with the properties of the onginal protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions
• the table below shows amino acids which may be substituted for an onginal amino acid in a protein and which are regarded as conservative amino acid substitutions Ongmal Residue Conservative Substitution
• Conservative ammo acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and or (c) the bulk of the side chain
• a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more ammo acid residues or nucleotides
• denvative refers to a chemically modified polynucleotide or polypeptide
• a denvative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule
• a denvative 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 de ⁇ ved
• a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide
• a “fragment” is a unique portion of SYNT or the polynucleotide encoding SYNT which is identical in sequence to but shorter in length than the parent sequence
• a fragment may comp ⁇ se up to the entire length of the defined sequence, minus one nucleotide/amino acid residue
• a fragment may comp ⁇ se from 5 to 1000 contiguous nucleotides or amino acid residues
• a fragment used as a probe, p ⁇ mer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60.
• Fragments may be preferentially selected from certain regions of a molecule
• a polypeptide fragment may comp ⁇ se a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50% of a polypeptide) as shown in a certain defined sequence
• these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments
• a fragment of SEQ ID NO 16-30 compnses a region of unique polynucleotide sequence that specifically identifies SEQ ID NO 16-30, for example, as distinct from any other sequence in the genome from which the fragment was obtained
• a fragment of SEQ ID NO 16-30 is useful, for example, in hybndization and amplification technologies and in analogous methods that distinguish SEQ ID NO 16-30 from related polynucleotide sequences
• the precise length of a fragment of SEQ ID NO 16-30 and the region of SEQ ID NO 16-30 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment
• a fragment of SEQ ID NO 1- 15 is encoded by a fragment of SEQ ID NO 16-30
• a fragment of SEQ ID NO 1-15 compnses a region of unique amino acid sequence that specifically identifies SEQ ID NO 1-15
• a fragment of SEQ ID NO 1- 15 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO
• a “full-length” polynucleotide sequence is one containing at least a translation initiation codon (e g , methionine) followed by an open reading frame and a translation termination codon
• a “full-length” polynucleotide sequence encodes a "full-length” polypeptide sequence
• “Homology” refers to sequence sinulanty or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences
• percent identity and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algonthm Such an algo ⁇ thm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful compa ⁇ son of the two sequences
• Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algonthm as incorporated into the MEGALIGN version 3 12e sequence alignment program This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI) CLUSTAL V is descnbed in Higgins, D G and P M Sharp ( 1989) CABIOS 5 151-153 and in Higgins, D G et al (1992) CABIOS 8 189-191 For pairwise alignments of polynucleotide sequences, the default parameters are set as follows.
• Biol 215:403-410 which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http://www.ncbi nlm.mh gov/BLAST/ The BLAST software suite includes vanous sequence analysis programs including "blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a va ⁇ ety of databases.
• BLAST 2 Sequences are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2.0.12 (Ap ⁇ l-21-2000) set at default parameters. Such default parameters may be, for example-
• Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
• Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
• Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
• the phrases "percent identity” and "% identity,” as applied to polypeptide sequences refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algonthm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
• NCBI BLAST software suite may be used.
• BLAST 2 Sequences Version 2.0.12 (Apr-21-2000) with blastp set at default parameters.
• Such default parameters may be, for example.
• Gap x drop-off 50
• Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
• Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to desc ⁇ be a length over which percentage identity may be measured.
• 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 chromosome replication, segregation and maintenance
• humanized antibody refers to an antibody molecule 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 onginal binding ability
• “Hybndization” refers to the process by which a polynucleotide strand anneals with a complementary strand through base painng under defined hybndization conditions
• Specific hybndization is an indication that two nucleic acid sequences share a high degree of complementarity Specific hybndization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s)
• the washing step(s) is particularly important in determining the stnngency of the hybndization process with more stnngent conditions allowing less non-specific binding, 1 e , binding between pairs of nucleic acid strands that are not perfectly matched Permissive conditions for annealing of nucleic acid sequences are routinely determinable
• stnngency of hybndization is expressed, in part, with reference to the temperature under which the wash step is earned out
• wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH
• T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe
• High stnngency conditions for hybndization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0 2 x SSC and about 0 1 % SDS, for 1 hour Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used SSC concentration may be varied from about 0 1 to 2 x SSC, with SDS being present at about 0 1 %
• blocking reagents are used to block non-specific hybndization
• Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA DNA hyb ⁇ dizations Useful vanations on these wash conditions will be readily apparent to those of ordinary skill in the art Hybndization, particularly under high stnngency conditions, may be suggestive
• hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases
• a hybridization complex may be formed in solution (e g , C 0 t or R ⁇ analysis) or formed between one nucleic acid sequence present m solution and another nucleic acid sequence immobilized on a solid support (e g , paper, membranes, filters, chips, pins or glass slides, or any other appropnate substrate to which cells or their nucleic acids have been fixed)
• insertion and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively
• 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 va ⁇ ous factors, e g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems
• an “immunogenic fragment” is a polypeptide or ohgopeptide fragment of SYNT which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal
• immunogenic fragment also includes any polypeptide or ohgopeptide fragment of SYNT which is useful in any of the antibody production methods disclosed herein or known in the art.
• microarray refers to an arrangement of a plurality of polynucleotides, polypeptides, or other chemical compounds on a substrate
• element and “array element” refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray
• nucleic acid and “nucleic acid sequence” refer to a nucleotide, ohgonucleotide, polynucleotide, or any fragment thereof These phrases also refer to DNA or RNA of genomic or synthetic ongin 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-hke or RNA-hke mate ⁇ al
• operably linked refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence
• a promoter is operably linked to a coding sequence if the promoter affects the transcnption or expression of the coding sequence.
• Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame
• PNA protein nucleic acid
• PNA refers to an antisense molecule or anti-gene agent which compnses an ohgonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of ammo acid residues ending m lysine.
• the terminal lysme confers solubility to the composition PNAs preferentially bind complementary single stranded DNA or RNA and stop transcnpt elongation, and may be pegylated to extend their hfespan in the cell
• Post-translational modification of an SYNT may involve hpidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically Biochemical modifications will vary by cell type depending on the enzymatic milieu of SYNT
• Probe refers to nucleic acid sequences encoding SYNT, their complements, or fragments thereof, which are used to detect identical, allehc or related nucleic acid sequences
• Probes are isolated ohgonucleotides or polynucleotides attached to a detectable label or reporter molecule Typical labels include radioactive isotopes, hgands, chemiluminescent agents, and enzymes
• P ⁇ mers are short nucleic acids, usually DNA ohgonucleotides, which may be annealed to a target polynucleotide by complementary base-painng The primer may then be extended along the target DNA strand by a DNA polymerase enzyme Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) Probes and p ⁇ mers as used in the present invention typically comp ⁇ se at least 15 contiguous nucleotides of a known sequence.
• PCR polymerase chain reaction
• probes and pnmers may also be employed, such as probes and p ⁇ mers that comp ⁇ se at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and p ⁇ mers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used
• PCR p ⁇ mer pairs can be de ⁇ ved from a known sequence, for example, by using computer programs intended for that purpose such as P ⁇ mer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambndge MA).
• O gonucleotides for use as p ⁇ mers are selected using software known in the art for such purpose. For example, OLIGO 4 06 software is useful for the selection of PCR pnmer pairs of up to 100 nucleotides each, and for the analysis of ohgonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabilities.
• the P ⁇ mOU pnmer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific pnmers from megabase sequences and is thus useful for designing pnmers on a genome-wide scope.
• the P ⁇ mer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambndge MA) allows the user to input a "misp ⁇ ming library," m which sequences to avoid as p ⁇ mer binding sites are user-specified.
• P ⁇ mer3 is useful, in particular, for the selection of o gonucleotides for microarrays.
• the source code for the latter two p ⁇ mer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs
• the P ⁇ meGen program available to the public from the UK Human Genome Mapping Project Resource Centre, Cambndge UK) designs p ⁇ mers based on multiple sequence alignments, thereby allowing selection of p ⁇ mers that hybndize to either the most conserved or least conserved regions of aligned nucleic acid sequences Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments
• the ohgonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing p ⁇ mers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids Methods of o gonucleotide selection are not limited to those described above A "recombinant nucle
• such recombinant nucleic acids may be part of a viral vector, e g , based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal
• a “regulatory element” refers to a nucleic acid sequence usually denved from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs) Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability
• Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, am o acid, or antibody Reporter molecules include radionuc des, enzymes, fluorescent, chemiluminescent, or chromogemc agents, substrates, cofactors, inhibitors, magnetic particles, and other moieties known in the art
• An "RNA equivalent,” in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of nbose instead of deoxy ⁇ bose
• sample is used in its broadest sense
• binding and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition The interaction is dependent upon the presence of a particular structure of the protein, e g , the antigenic determinant or epitope, recognized by the binding molecule For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody
• substantially pu ⁇ fied refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated
• Substrate refers to any suitable ngid or semi-ngid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillanes
• the substrate can have a va ⁇ ety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound
• a “transcript image” refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time
• Transformation desc ⁇ bes a process by which exogenous DNA is introduced into a recipient cell Transformation may occur under natural or artificial conditions according to va ⁇ ous 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, bactenophage or viral infection, electroporation, heat shock, hpofection, 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 pe ⁇ ods of time
• a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, m which one or more of the cells of the organism contains heterologous nucleic acid introduced by way
• a “variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2 0 9 (May-07- 1999) set at default parameters
• Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% or greater sequence identity over a certain defined length
• a vanant may be descnbed as, for example, an "allehc” (as defined above), "splice,” “species,” or “polymorphic” vanant
• a splice vanant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternative splicing of exons du ⁇ ng mRNA processing The corresponding polypeptide may
• a “vanant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2 0 9 (May-07- 1999) set at default parameters
• Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% or greater sequence identity over a certain defined length of one of the polypeptides THE INVENTION
• the invention is based on the discovery of new human synthetases (SYNT), the polynucleotides encoding SYNT, and the use of these compositions for the diagnosis, treatment, or prevention of immune, neuronal, and reproductive disorders, and cell prohferative disorders including cancer
• Table 1 lists the Incyte clones used to assemble full length nucleotide sequences encoding
• SYNT 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 SYNT were identified
• column 4 shows the cDNA hbranes 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 de ⁇ ved from pooled cDNA hbranes
• GenBank sequence identifiers are also shown in column 5.
• the Incyte clones and GenBank cDNA sequences, where indicated, in column 5 were used to assemble the consensus nucleotide sequence of each SYNT 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 compnsing signature sequences and motifs;
• column 6 shows homologous sequences as identified by BLAST analysis, and
• column 7 shows analytical methods and in some cases, searchable databases to which the analytical methods were applied The methods of column 7 were used to characte ⁇ ze each polypeptide through sequence homology and protein motifs.
• the columns of Table 3 show the tissue-specificity and diseases, disorders, or conditions associated with nucleotide sequences encoding SYNT.
• 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 hybndization or amplification technologies to identify 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 N0 23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO.29, and SEQ ID NO 30 and to distinguish between 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:25, SEQ ID NO:26, S
• Column 3 lists tissue categories which express SYNT as a fraction of total tissues expressing SYNT
• Column 4 lists diseases, disorders, or conditions associated with those tissues expressing SYNT as a fraction of total tissues expressing SYNT.
• Column 5 lists the vectors used to subclone each cDNA library The columns of Table 4 show descnptions of the tissues used to construct the cDNA hbranes from which cDNA clones encoding SYNT were isolated.
• Column 1 references the nucleotide SEQ ID NOs
• column 2 shows the cDNA hbranes from which these clones were isolated
• column 3 shows the tissue origins and other descriptive information relevant to the cDNA hbranes m column 2.
• SEQ ID NO 16 maps to chromosome 5 within the interval from 147 10 to 150.00 centiMorgans
• SEQ ID NO: 17 maps to chromosome 10 within the interval from 137.60 to 139.20 centiMorgans This interval also contains gene MXI1, a member of the MYC family.
• SEQ ID NO 18 maps to chromosome 2 within the interval from 228.80 to 230.10 centiMorgans This interval also contains a gene for a proto-oncogene encoding a tyrosine-protem kinase
• SEQ ID NO 21 maps to chromosome 5 within the interval from 172.6 to 184 7 centiMorgans
• SEQ ID NO'24 maps to chromosome 2 within the interval from 1 18 0 to 127.4 centiMorgans.
• SEQ ID NO 26 maps to chromosome 3 within the interval from 157 4 to 162.0 centiMorgans.
• SEQ ID NO 27 maps to chromosome 12 within the interval from 97 1 to 1 16.6 centiMorgans.
• SEQ ID NO 28 maps to chromosome 4 within the interval from 77 3 to 99.2 centiMorgans and to chromosome 5 within the intervals from 79.2 to 92.3 centiMorgans, from 1 16.3 to 127.9 centiMorgans, and from 157.6 to 163 0 centiMorgans
• SEQ ID NO:29 maps to chromosome 1 within the interval from 242.5 to 258.7 centiMorgans and to chromosome 19 within the interval from 69.9 to 104.9 centiMorgans.
• SEQ ID NO:30 maps to chromosome 1 within the interval from 57.2 to 57.5 centiMorgans.
• a preferred SYNT vanant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% ammo acid sequence identity to the SYNT ammo acid sequence, and which contains at least one functional or structural charactenstic of SYNT
• the invention also encompasses polynucleotides which encode SYNT.
• the invention encompasses a polynucleotide sequence compnsing a sequence selected from the group consisting of SEQ ID NO: 16-30, which encodes SYNT.
• the polynucleotide sequences of SEQ ID NO: 16-30 as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ⁇ bose instead of deoxynbose.
• the invention also encompasses a vanant of a polynucleotide sequence encoding SYNT.
• a vanant polynucleotide sequence will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding SYNT.
• a particular aspect of the invention encompasses a variant of a polynucleotide sequence compnsing a sequence selected from the group consisting of SEQ ID NO: 16-30 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 16-30.
• Any one of the polynucleotide va ⁇ ants desc ⁇ bed above can encode an amino acid sequence which contains at least one functional or structural characte ⁇ stic of SYNT.
• nucleotide sequences which encode SYNT and its vanants are generally capable of hybndizing to the nucleotide sequence of the naturally occumng SYNT under appropnately selected conditions of st ⁇ ngency, it may be advantageous to produce nucleotide sequences encoding SYNT or its de ⁇ vatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occumng codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
• RNA transc ⁇ pts having more desirable properties, such as a greater half-life, than transcnpts produced from the naturally occumng sequence
• the invention also encompasses production of DNA sequences which encode SYNT and SYNT denvatives, or fragments thereof, entirely by synthetic chemistry.
• the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art.
• synthetic chemistry may be used to introduce mutations into a sequence encoding SYNT or any fragment thereof.
• polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO: 16-30 and fragments thereof under various conditions of stnngency.
• Hybridization conditions including annealing and wash conditions, are described in "Definitions.”
• sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (PE Biosystems).
• Sequencing is then earned out using either the ABI 373 or 377 DNA sequencing system (PE Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art
• the resulting sequences are analyzed using a vanety of algonthms which are well known in the art. (See, e.g.,
• the nucleic acid sequences encoding SYNT may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements
• restriction-site PCR uses universal and nested pnmers to amplify unknown sequence from genomic DNA within a cloning vector (See, e g , Sarkar, G ( 1993) PCR Methods Apphc 2 318-322 )
• Another method, inverse PCR uses primers that extend in divergent directions to amplify unknown sequence from a circula ⁇ zed template The template is denved from restnction fragments compnsing a known genomic locus and surrounding sequences (See, e g , Tngha, T et al ( 1988) Nucleic Acids Res 16 8186 )
• capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA (See, e g , Tngha
• hbranes When screening for full-length cDNAs, it is preferable to use hbranes that have been size-selected to include larger cDNAs.
• random-p ⁇ med hbranes which often include sequences containing the 5' regions of genes, are preferable for situations in which an ohgo d(T) library does not yield a full-length cDNA Genomic hbranes may be useful for extension of sequence into 5' non-transcnbed regulatory regions
• Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products
• capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths
• Output light intensity may be converted to elect ⁇ cal signal using appropnate software (e g , GENOTYPER and SEQUENCE NAVIGATOR, PE Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled
• Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample
• polynucleotide sequences or fragments thereof which encode SYNT may be cloned in recombinant DNA molecules that direct expression of SYNT, or fragments or functional equivalents thereof, in appropnate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express SYNT
• nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter SYNT-encoding sequences for a vanety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic ohgonucleotides may be used to engineer the nucleotide sequences.
• ohgonucleotide- mediated site-directed mutagenesis may be used to introduce mutations that create new rest ⁇ ction sites, alter glycosylation patterns, change codon preference, produce splice vanants, and so forth
• the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA: desc ⁇ bed in U.S Patent Number 5,837,458, Chang, C -C. et al ( 1999) Nat. Biotechnol. 17:793-797; Ch ⁇ stians, F.C. et al. (1999) Nat. Biotechnol. 17.259-264; and Crame ⁇ , A. et al. ( 1996) Nat. Biotechnol 14-315-319) to alter or improve the biological properties of SYNT, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds.
• MOLECULARBREEDING Maxygen Inc., Santa Clara CA: desc ⁇ bed in U.S Patent Number 5,837,458, Chang, C -C. et al ( 1999) Nat. Biotechnol. 17:793-797; Ch ⁇ stians, F
• DNA shuffling is a process by which a library of gene va ⁇ ants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene vanants with the desired properties. These preferred vanants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
• genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized.
• fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occumng genes in a directed and controllable manner.
• sequences encoding SYNT may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser 7.215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7.225-232 )
• SYNT itself or a fragment thereof may be synthesized using chemical methods.
• peptide synthesis can be performed using vanous solution-phase or solid-phase techniques (See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Properties. WH Freeman, New York NY, pp 55-60; and Roberge, J Y.
• the nucleotide sequences encoding SYNT or de ⁇ vatives thereof may be inserted into an appropnate expression vector, I e., a vector which contains the necessary elements for transcnptional and translational control of the inserted coding sequence in a suitable host
• appropnate expression vector I e., a vector which contains the necessary elements for transcnptional and translational control of the inserted coding sequence in a suitable host
• These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5 ' and 3' untranslated regions in the vector and in polynucleotide sequences encoding SYNT
• Such elements may vary in their strength and specificity Specific initiation signals may also be used to achieve more efficient translation of sequences encoding SYNT.
• Such signals include the ATG initiation codon and adjacent sequences, e.g the Kozak sequence.
• sequences encoding SYNT and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcnptional or translational control signals may be needed
• exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector.
• Exogenous translational elements and initiation codons may be of vanous o ⁇ gins, both natural and synthetic The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used (See, e.g., Scharf, D et al ( 1994) Results Probl.
• a va ⁇ ety of expression vector/host systems may be utilized to contain and express sequences encoding SYNT
• microorganisms such as bactena transformed with recombinant bactenophage, 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 bacte ⁇ al expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
• microorganisms such as bactena transformed with recombinant bactenophage, 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, Ca
• Expression vectors denved from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from va ⁇ ous bactenal plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population.
• 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 SYNT.
• routine cloning, subcloning, and propagation of polynucleotide sequences encoding SYNT 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 SYNT into the vector's multiple cloning site disrupts the lac gene, allowing a colo ⁇ met ⁇ c screening procedure for identification of transformed bactena 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 SYNT may be used.
• vectors containing the strong, inducible T5 or T7 bactenophage promoter may be used.
• Yeast expression systems may be used for production of SYNT.
• a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomvces cerevisiae or Pichia pastons.
• 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 SYNT.
• Transcnption of sequences encoding SYNT may be d ⁇ ven 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 6:307-31 1).
• plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used.
• These 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 )
• a number of viral-based expression systems may be utilized.
• sequences encoding SYNT may be gated into an adenovirus transcnption/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses SYNT in host cells.
• transcnption 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
• HACs Human artificial chromosomes
• 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, polycatio c amino polymers, or vesicles) for therapeutic purposes.
• liposomes, polycatio c amino polymers, or vesicles for therapeutic purposes.
• stable expression of SYNT in cell lines is preferred.
• sequences encoding SYNT can be transformed into cell lines using expression vectors which may contain viral ongins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector.
• cells may be allowed to grow for about 1 to 2 days ennched media before being switched to selective media.
• the purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
• Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropnate to the cell type.
• any number of selection systems may be used to recover transformed cell lines
• These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphonbosyltransferase genes, for use in tk and apr cells, respectively (See, e.g., Wigler, M. et al. (1977) Cell 1 1 :223-232, Lowy, I. et al. (1980) Cell 22.817-823 )
• antimetabohte, antibiotic, or herbicide resistance can be used as the basis for selection.
• dhfr confers resistance to methotrexate
• neo confers resistance to the aminoglycosides neomycm and G-418
• als and pat confer resistance to chlorsulfuron and phosphinotncin acetyltransferase, respectively.
• Visible markers e.g., anthocyamns, green fluorescent proteins (GFP; Clontech), ⁇ glucuromdase and its substrate ⁇ -glucuronide, or luciferase and its substrate lucife ⁇ n may be used. These markers can be used not only to identify transformants. but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (See. e g., Rhodes, CA. ( 1995) Methods Mol. Biol. 55 121-131.)
• marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
• sequence encoding SYNT is inserted within a marker gene sequence
• transformed cells containing sequences encoding SYNT can be identified by the absence of marker gene function.
• a marker gene can be placed in tandem with a sequence encoding SYNT under the control of a single promoter Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well
• SYNT may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybndizations, PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. Immunological methods for detecting and measuring the expression of SYNT using either specific polyclonal or monoclonal antibodies are known in the art.
• ELISAs enzyme-linked immunosorbent assays
• RIAs radioimmunoassays
• FACS fluorescence activated cell sorting
• va ⁇ ety of labels and conjugation techniques are known by those skilled in the art and may be used in va ⁇ ous nucleic acid and amino acid assays.
• Means for producing labeled hybndization or PCR probes for detecting sequences related to polynucleotides encoding SYNT include oligolabehng, nick translation, end-labeling, or PCR amplification using a labeled nucleotide
• the sequences encoding SYNT, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
• RNA probes are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropnate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides
• an appropnate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides
• Host cells transformed with nucleotide sequences encoding SYNT 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 SYNT may be designed to contain signal sequences which direct secretion of SYNT 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
• modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, hpidation, and acylation
• Post-translational processing which cleaves a "prepro” or "pro” 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 charactenstic 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
• natural, modified, or recombinant nucleic acid sequences encoding SYNT may be hgated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
• a chimeric SYNT protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide hbranes for inhibitors of SYNT activity.
• Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matnces.
• Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-H ⁇ s, FLAG, c-myc, and hemagglutimn (HA) GST, MBP, Trx, CBP, and 6-H ⁇ s enable punfication of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
• GST glutathione S-transferase
• MBP maltose binding protein
• Trx thioredoxin
• CBP calmodulin binding peptide
• 6-H ⁇ s 6-H ⁇ s
• FLAG c-myc
• hemagglutimn (HA) GST, MBP, Trx, CBP, and 6-H ⁇ s enable punfication of their cognate fusion proteins on
• FLAG, c-myc, and hemagglutimn (HA) enable immunoaffinity punfication 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 SYNT encoding sequence and the heterologous protein sequence, so that SYNT may be cleaved away from the heterologous moiety following purification Methods for fusion protein expression and punfication are discussed in Ausubel ( 1995, supra, ch. 10).
• a va ⁇ ety of commercially available kits may also be used to facilitate expression and punfication of fusion proteins.
• synthesis of radiolabeled SYNT may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcnption and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S-meth ⁇ on ⁇ ne.
• SYNT of the present invention or fragments thereof may be used to screen for compounds that specifically bind to SYNT.
• At least one and up to a plurality of test compounds may be screened for specific binding to SYNT.
• Examples of test compounds include antibodies, o gonucleotides, proteins (e.g., receptors), or small molecules
• the compound thus identified is closely related to the natural ligand of SYNT, e.g , a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (See, Cohgan, J.E. et al. ( 1991) Current Protocols in Immunology 1(2) Chapter 5.)
• the compound can be closely related to the natural receptor to which SYNT binds, or to at least a fragment of the receptor, e.g., the ligand binding site.
• screening for these compounds involves producing appropnate cells which express SYNT, either as a secreted protein or on the cell membrane Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing SYNT or cell membrane fractions which contain SYNT are then contacted with a test compound and binding, stimulation, or inhibition of activity of either SYNT or the compound is analyzed.
• An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label.
• the assay may comp ⁇ se the steps of combining at least one test compound with SYNT, either in solution or affixed to a solid support, and detecting the binding of SYNT to the compound.
• the assay may detect or measure binding of a test compound in the presence of a labeled competitor.
• the assay may be earned out using cell-free preparations, chemical hbranes, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a solid support.
• SYNT of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of SYNT.
• Such compounds may include agonists, antagonists, or partial or inverse agonists.
• an assay is performed under conditions permissive for SYNT activity, wherein SYNT is combined with at least one test compound, and the activity of SYNT in the presence of a test compound is compared with the activity of SYNT in the absence of the test compound.
• a change in the activity of SYNT in the presence of the test compound is indicative of a compound that modulates the activity of SYNT
• a test compound is combined with an in vitro or cell-free system compnsing SYNT under conditions suitable for SYNT activity, and the assay is performed In either of these assays, a test compound which modulates the activity of SYNT may do so indirectly and need not come in direct contact with the test compound At least one and up to a plurality of test compounds may be screened
• polynucleotides encoding SYNT or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells
• ES embryonic stem
• Such techniques are well known in the art and are useful for the generation of animal models of human disease (See, e g , U S Patent No 5, 175,383 and U S Patent No 5,767,337 )
• mouse ES cells such as the mouse 129/SvJ cell line, are de ⁇ ved from the early mouse embryo and grown m culture
• the ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e g , the neomycin phosphotransferase gene (neo, Capecchi, M R ( 1989) Science 244 1288-1292)
• the vector integrates into the corresponding region of the host genome by homologous recombination Alternatively, homologous recombination takes place using the
• Polynucleotides encoding SYNT may also be manipulated in vitro in ES cells derived from human blastocysts Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J A et al (1998) Science 282 1 145-1 147)
• Polynucleotides encoding SYNT can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease
• knockin technology a region of a polynucleotide encoding SYNT is injected into animal ES cells, and the injected sequence integrates into the animal cell genome Transformed cells are injected into blastulae, and the blastulae are implanted as descnbed above Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease
• a mammal inbred to overexpress SYNT, e g by secreting SYNT in its milk, may also serve as a convenient source of that protein (Janne, J et al ( 1998) Biotechnol Annu Rev 4 55-74) THERAPEUTICS
• SYNT appears to play a role in an immune disorder such as inflammation, actinic keratosis, acquired immunodeficiency syndrome (AIDS), Addison s disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes melhtus, emphysema,
• an immune disorder such as inflammation, actinic keratosis, acquired immunodeficiency syndrome (AIDS), Addison s disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis
• a vector capable of expressing SYNT or a fragment or denvative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of SYNT including, but not limited to, those described above
• a pharmaceutical composition comprising a substantially punfied
• SYNT in conjunction with a suitable pharmaceutical earner may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of SYNT including, but not limited to, those provided above
• an agonist which modulates the activity of SYNT may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of SYNT including, but not limited to, those listed above
• an antagonist of SYNT may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of SYNT Examples of such disorders include, but are not limited to, those immune, neuronal, reproductive, and cell prohferative disorders descnbed above
• an antibody which specifically binds SYNT may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bnnging a pharmaceutical agent to cells or tissues which express SYNT
• a vector expressing the complement of the polynucleotide encoding SYNT may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of SYNT including, but not limited to, those descnbed above
• any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered m combination with other appropriate therapeutic agents
• Selection of the appropnate 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 vanous disorders desc ⁇ bed 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
• An antagonist of SYNT may be produced using methods which are generally known in the art
• punfied SYNT may be used to produce antibodies or to screen hbranes of pharmaceutical agents to identify those which specifically bind SYNT
• Antibodies to SYNT may also be generated using methods that are well known in the art
• Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimenc, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library
• Neutralizing antibodies I e , those which inhibit dimer formation
• va ⁇ ous hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with SYNT or with any fragment or ohgopeptide thereof which has immunogenic properties
• various adjuvants may be used to increase immunological response.
• Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
• BCG Bacilli Calmette-Guenn
• Corynebacte ⁇ um parvum are especially preferable
• the ohgopeptides, peptides, or fragments used to induce antibodies to SYNT have an ammo acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids.
• these ohgopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein Short stretches of SYNT ammo acids may be fused with those of another protein, such as KLH, and antibodies to the chimenc molecule may be produced
• Monoclonal antibodies to SYNT may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybndoma technique, the human B-cell hyb ⁇ doma technique, and the EBV-hyb ⁇ doma technique (See, e.g., Kohler, G. et al. (1975) Nature 256 495-497; Kozbor, D. et al. ( 1985) J
• chimenc antibodies such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used.
• Antibodies may also be produced by inducing in vivo production m the lymphocyte population or by screening immunoglobulin hbranes or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al (1989) Proc Natl. Acad. Sci USA 86:3833-3837; Winter, G. et al. ( 1991) Nature 349:293-299 )
• Antibody fragments which contain specific binding sites for SYNT may also be generated
• such fragments include, but are not limited to, F(ab') 2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bndges of the F(ab')2 fragments.
• Fab expression hbranes may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e g , Huse, W D et al.
• K a which is defined as the molar concentration of SYNT-antibody complex divided by the molar concentrations of free antigen and free antibody under equihb ⁇ um conditions
• K a association constant
• the K a determined for a preparation of monoclonal antibodies, which are monospecific for a particular SYNT epitope represents a true measure of affinity High-affinity antibody preparations with K, ranging from about 10 9 to 10 12 L/mole are preferred for use in immunoassays in which the SYNT-antibody complex must withstand ⁇ gorous manipulations
• Low-affinity antibody preparations with K a ranging from about 10 6 to 10 7 L/
• 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 generally employed in procedures requi ⁇ ng precipitation of SYNT-antibody complexes
• Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in vanous applications are generally available (See, e g , Catty, supra, and Cohgan et al , supra )
• the polynucleotides encoding SYNT, or any fragment or complement thereof may be used for therapeutic purposes
• modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified ohgonucleotides) to the coding or regulatory regions of the gene encoding SYNT
• DNA, RNA, PNA, or modified ohgonucleotides DNA, RNA, P
• Antisense sequences can be delivered mtracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein.
• Antisense sequences can also be introduced mtracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (See, e.g., Miller, A.D ( 1990) Blood 76.271. Ausubel, supra; Uckert, W. and W Walther ( 1994) Pharmacol. Ther 63(3):323-347.)
• viral vectors such as retrovirus and adeno-associated virus vectors
• Other gene delivery mechanisms include hposome-de ⁇ ved systems, artificial viral envelopes, and other systems known m the art (See, e.g., Rossi, J.J. ( 1995) Br. Med. Bull. 51(l):217-225; Boado, R.J. et al. ( 1998) J. Pharm. Sci. 87(1 1)- 1308-1315; and Moms, M.C. et al (1997) Nucleic Acids Res 25(14):2730-2736.)
• polynucleotides encoding SYNT may be used for somatic or germline gene therapy.
• Gene therapy may be performed to (l) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease characte ⁇ zed by X- hnked inhentance (Cavazzana-Calvo, M. et al. (2000) Science 288-669-672), severe combined immunodeficiency syndrome associated with an inhented adenosine deaminase (ADA) deficiency (Blaese, R M. et al. ( 1995) Science 270:475-480; Bordignon, C et al. ( 1995) Science 270 470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene
• conditionally lethal gene product e.g., in the case of cancers which result from unregulated cell proliferation
• a protein which affords protection against intracellular parasites e.g., against human retroviruses, such as human immunodeficiency virus (HIV) (Baltimore, D. ( 1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA.
• hepatitis B or C virus HBV, HCV
• fungal parasites such as Candida albicans and Paracoccidioides brasihensis, and protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi.
• the expression of SYNT from an appropnate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency
• diseases or disorders caused by deficiencies in SYNT are treated by constructing mammalian expression vectors encoding SYNT and introducing these vectors by mechanical means into SYNT-deficient cells.
• Mechanical transfer technologies for use with cells in vivo or ex vitro include (I) direct DNA micromjection into individual cells, (n) ballistic gold particle delivery, (in) hposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W F. Anderson ( 1993) Annu. Rev. Biochem. 62: 191-217; Ivies, Z. ( 1997) Cell 91 -501-510; Boulay, J-L. and H. Recipon ( 1998) Curr.
• Expression vectors that may be effective for the expression of SYNT include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
• SYNT may be expressed using (I) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (n) an inducible promoter (e.g , the tetracychne-regulated promoter (Gossen, M. and H. Bujard ( 1992) Proc. Natl. Acad. Sci. USA 89:5547-5551 ; Gossen, M. et al. ( 1995) Science 268.1766-1769; Rossi, F.M.V and H.M. Blau (1998) Curr. Opin. Biotechnol.
• a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes
• liposome transformation kits e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen
• PERFECT LIPID TRANSFECTION KIT available from Invitrogen
• transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb ( 1973) Virology 52.456-467), or by electroporation (Neumann, E. et al ( 1982) EMBO J. 1 :841-845).
• the introduction of DNA to pnmary cells requires modification of these standardized mammalian transfection protocols.
• diseases or disorders caused by genetic defects with respect to SYNT expression are treated by constructing a retrovirus vector consisting of (1) the polynucleotide encoding SYNT under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (n) appropnate RNA packaging signals, and (in) a Rev-responsive element (RRE) along with additional retrovirus cu-acting RNA sequences and coding sequences required for efficient vector propagation.
• Retrovirus vectors e.g., PFB and PFBNEO
• Retrovirus vectors are commercially available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc Natl. Acad. Sci.
• the vector is propagated an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al (1987) J. Virol. 61 : 1647-1650; Bender, M.A. et al. (1987) J. Virol. 61 : 1639-1646; Adam, M A. and A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. ( 1998) J Virol. 72-8463-8471 ; Zufferey, R et al. (1998) J.
• VPCL vector producing cell line
• U.S. Patent Number 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4 + T- cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U et al. ( 1997) J Virol. 71 :7020- 7029; Bauer, G. et al. ( 1997) Blood 89:2259-2267, Bonyhadi, ML.
• an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding SYNT to cells which have one or more genetic abnormalities with respect to the expression of SYNT.
• the construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E.
• adenoviral vectors are descnbed in U.S. Patent Number 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated by reference.
• Antmozzi P.A. et al. ( 1999) Annu. Rev. Nutr. 19:51 1-544; and Verma, I.M. and N. Somia ( 1997) Nature 18:389:239-242, both incorporated by reference herein.
• a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding SYNT to target cells which have one or more genetic abnormalities with respect to the expression of SYNT
• the use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing SYNT to cells of the central nervous system, for which HSV has a tropism.
• the construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art.
• a replication-competent herpes simplex virus (HSV) type 1 -based vector has been used to deliver a reporter gene to the eyes of p ⁇ mates (Liu, X. et al. (1999) Exp. Eye Res.169:385-395).
• HSV-1 virus vector has also been disclosed m detail in U.S Patent Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference.
• U.S. Patent Number 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropnate promoter for purposes including human gene therapy
• recombinant HSV strains deleted for ICP4, ICP27 and ICP22 are also taught by this patent.
• For HSV vectors see also Goins, W F. et al. ( 1999) J Virol.
• an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding SYNT to target cells
• SFV Semliki Forest Virus
• RNA replication a subgenomic RNA is generated that normally encodes the viral capsid proteins This subgenomic RNA replicates to higher levels than the full-length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e g., protease and polymerase) Similarly, inserting the coding sequence for SYNT into the alphavirus genome in place of the capsid-coding region results in the production of a large number of SYNT-codmg RNAs and the synthesis of high levels of SYNT in vector transduced cells.
• enzymatic activity e g., protease and polymerase
• alphavirus infection is typically associated with cell lysis within a few days, the ability to establish a persistent infection in hamster normal kidney cells (BHK-21) with a vanant of Smdbis virus (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A et al. (1997) Virology 228.74-83).
• the wide host range of alphaviruses will allow the introduction of SYNT into a vanety of cell types
• the specific transduction of a subset of cells in a population may require the sorting of cells p ⁇ or to transduction.
• the methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art
• Oligonucleotides de ⁇ ved from the transcnption initiation site may also be employed to inhibit gene expression.
• inhibition can be achieved using t ⁇ ple helix base-painng methodology T ⁇ ple helix pai ⁇ ng is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transc ⁇ ption factors, or regulatory molecules.
• t ⁇ plex DNA Recent therapeutic advances using t ⁇ plex DNA have been descnbed in the literature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches. Futura Publishing, Mt. Kisco NY, pp. 163- 177 )
• a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcnpt from binding to nbosomes.
• Ribozymes enzymatic RNA molecules
• Ribozymes may also be used to catalyze the specific cleavage of RNA
• the mechanism of ⁇ bozyme action involves sequence-specific hybndization of the nbozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
• engineered hammerhead motif nbozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding SYNT
• nbozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for nbozyme cleavage sites, including the following sequences GUA, GUU, and GUC Once identified, short RNA sequences of between 15 and 20 ⁇ bonucleotides, 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 hybndization with complementary o gonucleotides using nbonuclease protection assays
• RNA molecules may be generated by in vitro and in vivo transcnption of DNA sequences encoding SYNT Such DNA sequences may be incorporated into a wide vanety of vectors with suitable RNA polymerase promoters such as T7 or SP6 Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively 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
• An additional embodiment of the invention encompasses a method for screening for a compound which is effective m alternateng expression of a polynucleotide encoding SYNT
• Compounds which may be effective in alternativeng expression of a specific polynucleotide may include, but are not limited to, ohgonucleotides, antisense ohgonucleotides, tnple helix-forming ohgonucleotides, transc ⁇ ption factors and other polypeptide transcnptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences
• Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression
• a compound which specifically inhibits expression of the polynucleotide encoding SYNT may be therapeutically useful, and in the treament of disorders associated with decreased SYNT expression
• Alterations in the expression of a polynucleotide encoding SYNT are assayed by any method commonly known in the art Typically, the expression of a specific nucleotide is detected by hybndization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding SYNT The amount of hybndization may be quantified, thus forming the basis for a compa ⁇ son of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in alternativeng the expression of the polynucleotide.
• a screen for a compound effective in alternativeng expression of a specific polynucleotide can be earned out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D et al ( 1999) U.S. Patent No 5,932,435, Arndt, G.M. et al. (2000) Nucleic Acids Res 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al (2000) Biochem. Biophys Res Commun. 268 8- 13).
• a particular embodiment of the present invention involves screening a combinato ⁇ al library of ohgonucleotides (such as deoxynbonucleotides, ⁇ bonucleotides, peptide nucleic acids, and modified ohgonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T W et al. (1997) U.S. Patent No. 5.686,242; Bruice, T W et al (2000) U.S Patent No 6,022,691)
• ohgonucleotides such as deoxynbonucleotides, ⁇ bonucleotides, peptide nucleic acids, and modified ohgonucleotides
• vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. 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) Nat. Biotechnol 15-462-466 ) Any of the therapeutic methods descnbed above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys
• An additional embodiment of the invention relates to the administration of a pharmaceutical composition which generally compnses an active ingredient formulated with a pharmaceutically acceptable excipient
• Excipients may include, for example, sugars, starches, celluloses, gums, and proteins Va ⁇ ous formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA).
• Such pharmaceutical compositions may consist of SYNT, antibodies to SYNT, and mimetics, agonists, antagonists, or inhibitors of SYNT
• compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, lntra-arte ⁇ al, intramedullary, intrathecal, intravent ⁇ cular, pulmonary, transdermal, subcutaneous, intrapentoneal, intranasal, enteral, topical, sublingual, or rectal means.
• compositions for pulmonary administration may be prepared in liquid or dry powder form. These compositions are generally aerosolized immediately pnor to inhalation by the patient.
• aerosol delivery of fast-acting formulations is well-known in the art.
• macromolecules e g larger peptides and proteins
• Pulmonary delivery has the advantage of administration without needle injection, and obviates the need for potentially toxic penetration enhancers.
• compositions suitable for use in the invention include compositions wherein the active ingredients are contained m an effective amount to achieve the intended purpose The determination of an effective dose is well within the capability of those skilled in the art.
• Specialized forms of pharmaceutical compositions may be prepared for direct intracellular delivery of macromolecules compnsing SYNT or fragments thereof.
• liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule.
• SYNT or a fragment thereof may be joined to a short cationic N-terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285: 1569-1572).
• the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs.
• An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
• a therapeutically effective dose refers to that amount of active ingredient, for example SYNT or fragments thereof, antibodies of SYNT, and agonists, antagonists or inhibitors of SYNT, which ameliorates the symptoms or condition.
• Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with expe ⁇ mental 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 50 /ED, 0 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
• 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 seventy of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combmat ⁇ on(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
• Normal dosage amounts may vary from about 0 1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration
• Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc DIAGNOSTICS
• antibodies which specifically bind SYNT may be used for the diagnosis of disorders characte ⁇ zed by expression of SYNT, or in assays to monitor patients being treated with SYNT or agonists, antagonists, or inhibitors of SYNT
• Antibodies useful for diagnostic purposes may be prepared in the same manner as desc ⁇ bed above for therapeutics Diagnostic assays for SYNT include methods which utilize the antibody and a label to detect SYNT m human body fluids or m 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 va ⁇ ety of reporter molecules, several of which are descnbed above, are known in the art and may be used
• a variety of protocols for measunng SYNT including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of SYNT expression
• Normal or standard values for SYNT expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibody to SYNT under conditions suitable for complex formation
• the amount of standard complex formation may be quantitated by va ⁇ ous methods, such as photometnc means Quantities of SYNT 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 SYNT may be used for diagnostic purposes.
• the polynucleotides which may be used include ohgonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
• the polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of SYNT may be correlated with disease
• the diagnostic assay may be used to determine absence, presence, and excess expression of SYNT, and to monitor regulation of SYNT levels during therapeutic intervention
• hybndization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding SYNT or closely related molecules may be used to identify nucleic acid sequences which encode SYNT.
• 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 hybndization or amplification will determine whether the probe identifies only naturally occurring sequences encoding SYNT, allehc vanants, or related sequences
• Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the SYNT encoding sequences
• the hybridization probes of the subject invention may be DNA or RNA and may be denved from the sequence of SEQ ID NO- 16-30 or from genomic sequences including promoters, enhancers, and introns of the SYNT gene
• Means for producing specific hybndization probes for DNAs encoding SYNT include the cloning of polynucleotide sequences encoding SYNT or SYNT denvatives into vectors for the production of mRNA probes Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropnate RNA polymerases and the appropriate labeled nucleotides
• Hybndization probes may be labeled by a va ⁇ ety of reporter groups, for example, by radionuchdes such as 32 P or
• Polynucleotide sequences encoding SYNT may be used for the diagnosis of disorders associated with expression of SYNT
• disorders include, but are not limited to, an immune disorder such as inflammation, actinic keratosis, acquired immunodeficiency syndrome (AIDS), Addison 's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, arte ⁇ osclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes melhtus, emphysema, erythroblastosis fetahs, erythema nodosum, atrophic gast ⁇ tis, glomerulonephntis, Goodpasture's syndrome, gout,
• the nucleotide sequences encoding SYNT may be useful m assays that detect the presence of associated disorders, particularly those mentioned above
• the nucleotide sequences encoding SYNT may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybndization complexes After a suitable incubation penod, the sample is washed and the signal is quantified and compared with a standard value If the amount of signal m the patient sample is significantly altered in companson to a control sample then the presence of altered levels of nucleotide sequences encoding SYNT m the sample indicates the presence of the associated disorder.
• Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical tnals, or to monitor the treatment of an individual patient. In order to provide a basis for the diagnosis of a disorder associated with expression of
• SYNT a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding SYNT, under conditions suitable for hybndization or amplification. Standard hybndization may be quantified by companng the values obtained from normal subjects with values from an expenment in which a known amount of a substantially punfied polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder Deviation from standard values is used to establish the presence of a disorder
• hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject The results obtained from successive assays may be used to show the efficacy of treatment over a penod ranging from several days to months
• the presence of an abnormal amount of transcnpt (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease p ⁇ or to the appearance of actual clinical symptoms.
• a more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer
• Additional diagnostic uses for ohgonucleotides designed from the sequences encoding SYNT may involve the use of PCR.
• Ohgomers may be chemically synthesized, generated enzymatically, or produced m vitro
• Ohgomers will preferably contain a fragment of a polynucleotide encoding SYNT, or a fragment of a polynucleotide complementary to the polynucleotide encoding SYNT, and will be employed under optimized conditions for identification of a specific gene or condition.
• Ohgomers may also be employed under less stnngent conditions for detection or quantification of closely related DNA or RNA sequences
• ohgonucleotide pnmers derived from the polynucleotide sequences encoding SYNT may be used to detect single nucleotide polymorphisms (SNPs).
• SNPs are substitutions, insertions and deletions that are a frequent cause of inhe ⁇ ted or acquired genetic disease in humans
• Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods
• SSCP single-stranded conformation polymorphism
• fSSCP fluorescent SSCP
• ohgonucleotide p ⁇ mers denved from the polynucleotide sequences encoding SYNT are used to amplify DNA using the polymerase chain reaction (PCR)
• PCR polymerase chain reaction
• the DNA may be denved, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like SNPs in the DNA cause differences
• Methods which may also be used to quantify the expression of SYNT include radiolabelmg or biotinylatmg nucleotides, coamphfication of a control nucleic acid, and interpolating results from standard curves (See, e g , Melby, P C et al ( 1993) J Immunol.
• ohgonucleotides or longer fragments denved from any of the polynucleotide sequences descnbed herein may be used as elements on a microarray
• the microarray can be used in transcnpt imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as descnbed in Seilhamer, J J et al , "Comparative Gene Transcnpt Analysis," U S Patent No 5,840,484, incorporated herein by reference
• the microarray may also be used to identify genetic va ⁇ ants, mutations, and polymorphisms This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropnate and effective treatment regimen for that patient For example, therapeutic agents which are highly effective and display
• antibodies specific for SYNT, or SYNT or fragments thereof may be used as elements on a microarray
• the microarray may be used to monitor or measure protein-protem interactions, drug-target interactions, and gene expression profiles, as descnbed above
• a particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type.
• a transcript image represents the global pattern of gene expression by a particular tissue or cell type Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis," U.S.
• a transcnpt image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transc ⁇ pts or reverse transc ⁇ pts of a particular tissue or cell type.
• the hybndization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements compnse a subset of a plurality of elements on a microarray
• the resultant transcnpt image would provide a profile of gene activity.
• Transcript images may be generated using transc ⁇ pts isolated from tissues, cell lines, biopsies, or other biological samples
• the transcnpt image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or vitro, as m the case of a cell line
• Transcnpt images which profile the expression of the polynucleotides of the present invention may also be used m conjunction with in vitro model systems and prechnical evaluation of pharmaceuticals, as well as toxicological testing of indust ⁇ al and naturally-occurring environmental compounds All compounds induce charactenstic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al.
• the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound.
• Nucleic acids that are expressed in the treated biological sample are hyb ⁇ dized with one or more probes specific to the polynucleotides of the present invention, so that transcnpt levels corresponding to the polynucleotides of the present invention may be quantified
• the transcnpt levels in the treated biological sample are compared with levels in an untreated biological sample Differences in the transcnpt levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample
• proteome refers to the global pattern of protein expression in a particular tissue or cell type
• proteome expression patterns, or profiles are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time
• a profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type
• the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectnc focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Sterner and Anderson, supra)
• the proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent
• a proteomic profile may also be generated using antibodies specific for SYNT to quantify the levels of SYNT expression
• the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A et al ( 1999) Anal Biochem 270 103- 1 1 1 , Mendoze, L G et al (1999) Biotechniques 27 778-788)
• Detection may be performed by a va ⁇ ety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or ammo-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element
• Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcnpt level There is a poor correlation between transcnpt and protein abundances for some proteins in some tissues (Anderson,
• proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcnpt image, but which alter the proteomic profile.
• the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases
• the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
• the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample
• Microarrays may be prepared, used, and analyzed using methods known m the art.
• methods known m the art See, e.g , Brennan, T.M. et al. ( 1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl Acad Sci. USA 93: 10614-10619; Baldeschweiler et al. (1995) PCT application W095/251 1 16; Shalon, D et al ( 1995) PCT application WO95/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci USA 94:2150- 2155; and Heller, M.J. et al. (1997) U.S.
• Patent No. 5,605,662. Various types of microarrays are well known and thoroughly desc ⁇ bed in DNA Microarrays. A Practical Approach. M. Schena, ed. (1999) Oxford University Press, London, hereby expressly incorporated by reference.
• nucleic acid sequences encoding SYNT may be used to generate hybndization probes useful in mapping the naturally occumng genomic sequence
• Either coding or noncodmg sequences may be used, and in some instances, noncodmg sequences may be preferable over coding sequences.
• sequences of the invention 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), bactenal artificial chromosomes (BACs), bactenal PI constructions, or single chromosome cDNA libraries (See, e g , Har ⁇ ngton, J J et al ( 1997) Nat Genet 15 345-355 Pnce, C M ( 1993) Blood Rev 7 127-134, and Trask, B J ( 1991) Trends Genet 7 149-154 )
• the nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which correlate the mhentance of a disease state with the inheritance of
• Fluorescent in situ hybndization may be correlated with other physical and genetic map data (See, e g , Heinz-Ul ⁇ ch, et al ( 1995) in Meyers, supra, pp 965-968 ) Examples of genetic map data can be found m va ⁇ ous scientific journals or at the Online Mendehan Inhe ⁇ tance m Man (OMIM) World Wide Web site Correlation between the location of the gene encoding SYNT on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts
• SYNT in another embodiment, SYNT, its catalytic or immunogenic fragments, or ohgopeptides thereof can be used for screening hbranes of compounds in any of a vanety 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 mtracellularly
• the formation of binding complexes between SYNT 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 )
• This method large numbers of different small test compounds are synthesized on a solid substrate
• the test compounds are reacted with SYNT, or fragments thereof, and washed Bound SYNT is then detected by methods well known in the art Punfied SYNT can also be coated directly onto plates for use in the aforementioned drug screening techniques
• non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support
• the nucleotide sequences which encode SYNT may be used in any molecular
• RNA was purchased from Clontech or isolated from tissues described in Table 4 SoVne tissues were homogenized and lysed in guamdinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate The resulting lysates were centnfuged over CsCl cushions or extracted with chloroform RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA purity In some cases, RNA was treated with DNase For most hbranes, poly(A+) RNA was isolated using ohgo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA
• RNA was provided with RNA and constructed the corresponding cDNA hbranes
• cDNA was synthesized and cDNA hbranes 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 transc ⁇ ption was initiated using ohgo d(T) or random p ⁇ mers
• Synthetic ohgonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropnate restriction enzyme or enzymes. For most hbranes.
• cDNA was size-selected (300- 1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis.
• cDNAs were ligated into compatible rest ⁇ ction enzyme sites of the polyhnker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORTl plasmid (Life Technologies), pcDNA2.1 plasmid (Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Genomics, Palo Alto CA)
• a suitable plasmid e.g., PBLUESCRIPT plasmid (Stratagene), PSPORTl plasmid (Life Technologies), pcDNA2.1 plasmid (Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Genomics,
• Plasmids obtained as desc ⁇ bed in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were punfied using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Mi prep punfication kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid punfication systems or the R E.A.L. PREP 96 plasmid punfication kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophihzation, 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 fluoromet ⁇ cally using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland)
• Incyte cDNA recovered in plasmids as descnbed in Example II were sequenced as follows Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (PE Biosystems) 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 (PE Biosystems).
• Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were earned out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (PE Biosystems) 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 VI
• the polynucleotide sequences derived from cDNA sequencing were assembled and analyzed using a combination of software programs which utilize algonthms well known to those skilled in the art Table 5 summa ⁇ zes the tools, programs, and algonthms used and provides applicable descriptions, references, and threshold parameters
• Table 5 summa ⁇ zes the tools, programs, and algonthms used and provides applicable descriptions, references, and threshold parameters
• the first column of Table 5 shows the tools, programs, and algonthms used
• the second column provides bnef descriptions thereof
• the third column presents appropnate references, all of which are incorporated by reference herein in their entirety
• the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score, the greater the homology between two sequences)
• Sequences were analyzed using MACDNASIS PRO software (Hitachi Software Engineenng, South San Francisco CA) and LASERGENE software (DNASTAR)
• 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 que ⁇ ed against a selection of public databases such as the GenBank pnmate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM 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
• the full length polynucleotide sequences were translated to de ⁇ ve the corresponding full length ammo acid sequences, and these full length sequences were subsequently analyzed by query
• Northern analysis is a laboratory technique used to detect the presence of a transcnpt of a gene and involves the hybndization of a labeled nucleotide sequence to 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.)
• the product score takes into account both the degree of similanty between two sequences and the length of the sequence match.
• the product score is a normalized value between 0 and 100, and is calculated as follows- the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences).
• the BLAST score is calculated by assigning a score of +5 for every base that matches in a high-sco ⁇ ng segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score.
• the product score represents a balance between fractional overlap and quality in a BLAST alignment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap
• the results of northern analyses are reported as a percentage distnbution of hbranes in which the transcnpt encoding SYNT occurred.
• Analysis involved the catego ⁇ zation of cDNA hbranes by organ/tissue and disease.
• the organ/tissue categones included cardiovascular, dermatologic, developmental, endoc ⁇ ne, gastrointestinal, hematopoietic/immune, musculoskeletal, nervous, reproductive, and urologic.
• the disease/condition categones included cancer, inflammation, trauma, cell proliferation, neurological, and pooled.
• the full length nucleic acid sequences of SEQ ID NO: 16-30 were produced by extension of an appropnate fragment of the full length molecule using ohgonucleotide pnmers designed from this fragment.
• One pnmer was synthesized to initiate 5' extension of the known fragment, and the other primer, to initiate 3' extension of the known fragment
• the initial pnmers 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 72°C. Any stretch of nucleotides which would result in hairpin structures and p ⁇ mer-pnmer dime ⁇ zations was avoided
• PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.)
• the reaction mix contained DNA template, 200 nmol of each p ⁇ mer, reaction buffer containing Mg 2 *, (NH 4 ) 2 S0 4 , and ⁇ -mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for p ⁇ mer pair PCI A and PCI B: Step 1. 94°C, 3 mm; Step 2: 94°C, 15 sec; Step 3. 60°C, 1 mm; Step 4.
• Step 7 storage at 4°C
• the parameters for pnmer pair T7 and SK+ were as follows: Step 1 94°C, 3 mm, Step 2 94°C, 15 sec, Step 3 57 °C, 1 min; Step 4: 68°C, 2 mm; Step 5- Steps 2, 3, and 4 repeated 20 times, Step 6 68 °C, 5 min; Step 7 storage at 4°C
• the concentration of DNA in each well was determined by dispensing 100 ⁇ l PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE and 0.5 ⁇ l of undiluted PCR product into each well of an opaque fluonmeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent.
• 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 pnor to rehgation into pUC 18 vector (Amersham Pharmacia Biotech).
• CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
• sonicated or sheared pnor to rehgation into pUC 18 vector Amersham Pharmacia Biotech.
• 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 rehgated using T4 hgase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in rest ⁇ ction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 °C in 384-well plates in LB/2x carb liquid media
• Step 1 94°C, 3 m ; Step 2: 94°C 15 sec; Step 3 60°C, 1 min; Step 4. 72°C, 2 m , Step 5- steps 2, 3, and 4 repeated 29 times; Step 6- 72°C, 5 mm; Step 7- storage at 4°C DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above Samples with low DNA recovenes were reamp fied using the same conditions as described above.
• polynucleotide sequences of SEQ ID NO: 16-30 are used to obtain 5' regulatory sequences using the procedure above, along with ohgonucleotides designed for such extension, and an appropnate genomic library.
• the cDNA sequences which were used to assemble SEQ ID NO: 16-30 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith- Waterman algonthm. Sequences from these databases that matched SEQ ID NO: 16-30 were assembled into clusters of contiguous and overlapping sequences using assembly algonthms such as Phrap (Table 5) Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ ID NO-, to that map location.
• assembly algonthms such as Phrap (Table 5)
• Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR),
• SEQ ID NO.16, SEQ ID NO: 17, SEQ ID NO- 18, SEQ ID NO:21, SEQ ID NO.24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO-29, and SEQ ID NO'30 are desc ⁇ bed in The Invention as ranges, or intervals, of human chromosomes More than one map location is reported for SEQ ID NO.28 and SEQ ID NO:29, indicating that previously mapped sequences having similanty, but not complete identity, to SEQ ID N0 28 and SEQ ID NO:29 were assembled into their respective clusters.
• centiM centiMorgan
• centiM centiMorgan
• Mb megabase
• the cM distances are based on genetic markers mapped by Genethon which provide boundanes for radiation hybnd markers whose sequences were included in each of the clusters Diseases associated with the public and Incyte sequences located within the indicated intervals are also reported in the Invention where applicable.
• Hybndization probes de ⁇ ved from SEQ ID NO 16-30 are employed to screen cDNAs, genomic DNAs. or mRNAs
• ohgonucleotides consisting of about 20 base pairs
• OLIGO 4 06 software National Biosciences
• the labeled ohgonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech) An aliquot containing 10 7 counts per minute of the labeled probe is used
• the linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, piezoelectnc pnnting (ink-jet printing, See, e g , Baldeschweiler, supra), mechanical microspotting technologies, and denvatives thereof
• the substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena ( 1999), supra)
• Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers
• a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures
• a typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropnate number of elements (See, e g , Schena, M et al ( 1995) Science 270 467-470, Shalon, D et al ( 1996) Genome
• ESTs Expressed Sequence Tags
• fragments or ohgomers thereof may compnse the elements of the microarray Fragments or ohgomers suitable for hybndization can be selected using software well known in the art such as LASERGENE software (DNASTAR)
• the array elements are hybndized with polynucleotides in a biological sample
• the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection
• nonhybndized nucleotides from the biological sample are removed, and a fluorescence scanner is used to detect hybridization at each array element Alternatively, laser desorbtion and mass spectrometry may be used for detection of hybndization
• the degree of complementanty and the relative abundance of each polynucleotide which hybndizes to an element on the microarray may be assessed
• microarray preparation and usage is descnbed in detail below Tissue or Cell Sample
• RNA is isolated from tissue samples using the guamdinium thiocyanate method and poly(A) + RNA is punfied using the ohgo-(dT) cellulose method
• Each poly(A) + RNA sample is reverse transc ⁇ bed using MMLV reverse-transc ⁇ ptase, 0 05 pg/ ⁇ l ohgo-(dT) primer (21mer), IX first strand buffer, 0 03 units/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech)
• the reverse transcnption reaction is performed in a 25 ml volume containing 200 ng poly( A) + RNA with GEMBRIGHT kits (Incyte)
• Specific control poly(A) + RNAs are synthesized by in vitro trans
• Sequences of the present invention are used to generate array elements Each array element is amplified from bactenal cells containing vectors with cloned cDNA inserts PCR amplification uses p ⁇ mers complementary to the vector sequences flanking the cDNA insert Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g Amplified array elements are then punfied using SEPHACRYL-400 (Amersham Pharmacia Biotech) Punfied array elements are immobilized on polymer-coated glass slides Glass microscope slides (Corning) are cleaned by ultrasound in 0 1 % SDS and acetone, with extensive distilled water washes between and after treatments Glass slides are etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0 05% ammopropyl silane (Sigma) in 95% ethanol Coated slides are cured in a 1 10°C oven
• Array elements are applied to the coated glass substrate using a procedure described in US Patent No 5,807 522, incorporated herein by reference 1 ⁇ l of the array element DNA, at an average concentration of 100 ng/ ⁇ l, is loaded into the open capillary printing element by a high-speed robotic apparatus The apparatus then deposits about 5 nl of array element sample per slide
• Microarrays are UV-crosslinked using a STRATALINKER UV-crosshnker (Stratagene) Microarrays are washed at room temperature once in 0 2% SDS and three times in distilled water Non-specific binding sites are blocked by incubation of microarrays in 0 2% casein in phosphate buffered saline (PBS) (Tropix, Inc . Bedford MA) for 30 minutes at 60 °C followed by washes in 0 2% SDS and distilled water as before Hybridization Hybridization reactions contain 9 ⁇ l of sample mixture consisting of 0 2 ⁇ g each of Cy3 and
• Innova 70 mixed gas 10 W laser (Coherent, Inc , Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5
• the excitation laser light is focused on the array using a 20X microscope objective (Nikon, Inc , Melville NY)
• the slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster- scanned past the objective
• the 1 8 cm x 1 8 cm array used in the present example is scanned with a resolution of 20 micrometers
• a mixed gas multiline laser excites the two fluorophores sequentially Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, B ⁇ dgewater NJ) corresponding to the two fluorophores
• Appropnate filters positioned between the array and the photomultiplier tubes are used to filter the signals
• the emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5
• Each array is typically scanned twice, one scan per fluorophore using the appropnate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously
• the sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration
• a specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybndizing species of 1 100,000
• the output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc , Norwood MA) installed in an IBM-compatible PC computer
• A/D analog-to-digital
• the digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal)
• the data is also analyzed quantitatively Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore' s emission spectrum
• a g ⁇ d is supe ⁇ mposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid
• the fluorescence signal within each element is then integrated to obtain a numencal value corresponding to the average intensity of the signal
• the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte)
• Sequences complementary to the SYNT-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occumng SYNT
• ohgonucleotides compnsing from about 15 to 30 base pairs is descnbed, essentially the same procedure is used with smaller or with larger sequence fragments
• Appropnate ohgonucleotides are designed using OLIGO 4 06 software (National Biosciences) and the coding sequence of SYNT
• a complementary o gonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence
• a complementary ohgonucleotide is designed to prevent ⁇ bosomal binding to the SYNT-encod g transcnpt
• SYNT expression and punfication of SYNT is achieved using bacterial or virus-based expression systems
• cDNA is subcloned into an appropnate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcnption
• promoters include, but are not limited to, the trp-lac (tac) hyb ⁇ d promoter and the T5 or T7 bactenophage promoter in conjunction with the lac operator regulatory element
• Recombinant vectors are transformed into suitable bactenal hosts, e g , BL21(DE3)
• Antibiotic resistant bacteria express SYNT upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG)
• IPTG isopropyl beta-D- thiogalactopyranoside
• the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding SYNT by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
• Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculovirus. (See Engelhard, E.K. et al. ( 1994) Proc. Natl. Acad. Sci. USA 91 :3224-3227; Sandig, V. et al. ( 1996) Hum. Gene Ther. 7: 1937-1945.)
• SYNT is synthesized as a fusion protein with, e.g., glutathione S- transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates.
• GST a 26- kilodalton enzyme from Schistosoma iaponicum. enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST moiety can be proteolytically cleaved from SYNT at specifically engineered sites.
• 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 SYNT obtained by these methods can be used directly in the assays shown in Examples X and XIV.
• An SYNT activity assay measures aminoacylation of tRNA in the presence of a radiolabeled substrate.
• a cell-free extract depleted of endogenous aminoacyl-tRNA synthetase is prepared from Escherichia coli. SYNT, either biochemically purified or recombinantly produced, is added to the cell free extract. The cell-free extract is incubated with [ 14 C]-labeled amino acid under conditions favorable for translation. Incorporation of the [ 14 C]-labeled amino acid into acid-precipitable aminoacyl-tRNA is measured using a radioisotope counter. The amount of the [ 14 C]-labeled amino acid incorporated into aminoacyl tRNA is proportional to the amount of SYNT activity.
• SYNT activity may be assayed as follows. SYNT, or biologically active fragments thereof, are labeled with i25 I 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 SYNT, washed, and any wells with labeled SYNT complex are assayed. Data obtained using different concentrations of SYNT are used to calculate values for the number, affinity, and association of SYNT with the candidate molecules.
• SYNT function is assessed by expressing the sequences encoding SYNT at physiologically elevated levels in mammalian cell culture systems cDNA is subcloned into a mammalian expression vector containing a strong promoter that d ⁇ ves high levels of cDNA expression
• Vectors of choice include pCMV SPORT plasmid (Life Technologies) and pCR3 1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter 5-10 ⁇ g of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector Mark
• FCM Flow cytometry
• FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death
• events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide, changes in cell size and granula ⁇ ty as measured by forward light scatter and 90 degree side light scatter, down-regulation of DNA synthesis as measured by decrease in bromodeoxyu ⁇ dine uptake, alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies, and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface
• Methods in flow cytometry are discussed in Ormerod, M G ( 1994) Flow Cytometry. Oxford, New York NY
• SYNT The influence of SYNT on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding SYNT 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 SYNT and other genes of interest can be analyzed by northern analysis or microarray techniques XII. Production of SYNT Specific Antibodies
• the SYNT amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding ohgopeptide is synthesized and used to raise antibodies by means known to those of skill in the art Methods for selection of appropnate 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 1 1 )
• ohgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (PE Biosystems) using FMOC chemistry and coupled to KLH (Sigma-Ald ⁇ ch, St Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (See, e g , Ausubel, 1995, supra ) Rabbits are immunized with the ohgopeptide- KLH complex in complete Freund's adjuvant Resulting antisera are tested for antipeptide and anti- SYNT activity by, for example, binding the peptide or SYNT to a substrate, blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting with radio-iodmated goat anti-rabbit IgG XIII. Purification of Naturally Occurring SYNT Using Specific Antibodies
• Naturally occurring or recombinant SYNT is substantially purified by immunoaffinity chromatography using antibodies specific for SYNT
• An immunoaffinity column is constructed by covalently coupling anti-SYNT 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
• Media containing SYNT are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of SYNT (e g , high ionic strength buffers in the presence of detergent)
• the column is eluted under conditions that disrupt antibody/SYNT binding (e g , a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and SYNT is collected
• SYNT or biologically active fragments thereof, are labeled with I25 I Bolton-Hunter reagent (See, e g , Bolton A E and W M Hunter (1973) Biochem J 133 529-539 )
• I25 I Bolton-Hunter reagent See, e g , Bolton A E and W M Hunter (1973) Biochem J 133 529-539
• Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled SYNT, washed, and any wells with labeled SYNT complex are assayed
• Data obtained using different concentrations of SYNT are used to calculate values for the number, affinity, and association of SYNT with the candidate molecules
• molecules interacting with SYNT are analyzed using the yeast two-hybnd system as desc ⁇ bed in Fields, S and O Song (1989, Nature 340 245-246), or using commercially available kits based on the two-hybnd system, such as the MATCHMAKER system (Clontech)
• SYNT may also be used in the PATHCALLING process (CuraGen Corp , New Haven CT) which employs the yeast two-hybnd system in a high-throughput manner to determine all interactions between the proteins encoded by two large hbranes of genes (Nandabalan, K et al (2000) U S Patent No 6,057, 101 )
• ABI FACTURA A program that removes vector sequences and PE Biosystems, Foster City, CA. masks ambiguous bases in nucleic acid sequences.
• ABI/PARACEL FDF A Fast Data Finder useful in comparing and PE Biosystems, Foster City, CA; Mismatch ⁇ 50% annotating amino acid or nucleic acid sequences. Paracel Inc., Pasadena, CA.
• ABI AutoAssembler A program that assembles nucleic acid sequences. PE Biosystems, Foster City, CA.
• fastx score 100 or greater
• Phred A base-calling algorithm that examines automated Ewing, B. et al. (1998) Genome Res. sequencer traces with high sensitivity and 8: 175-185; Ewing, B. and P. Green probability. (1998) Genome Res. 8: 186-194.

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