US20020058264A1 - Human regulatory molecules - Google Patents

Human regulatory molecules Download PDF

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US20020058264A1
US20020058264A1 US09/840,787 US84078701A US2002058264A1 US 20020058264 A1 US20020058264 A1 US 20020058264A1 US 84078701 A US84078701 A US 84078701A US 2002058264 A1 US2002058264 A1 US 2002058264A1
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Preeti Lal
Jennifer Hillman
Olga Bandman
Purvi Shah
Janice Au-Young
Henry Yue
Karl Guegler
Neil Corley
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Incyte Corp
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Incyte Pharmaceuticals Inc
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Priority to US09/877,633 priority Critical patent/US20020102569A1/en
Application filed by Incyte Pharmaceuticals Inc filed Critical Incyte Pharmaceuticals Inc
Priority to US09/840,787 priority patent/US20020058264A1/en
Publication of US20020058264A1 publication Critical patent/US20020058264A1/en
Priority to US10/247,451 priority patent/US20040018188A9/en
Abandoned legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Definitions

  • This invention relates to nucleic acid and amino acid sequences of human regulatory molecules which are implicated in disease and to the use of these sequences in the diagnosis and treatment of diseases associated with cell proliferation.
  • Regulatory protein molecules function to control gene expression. These molecules turn individual or groups of genes on and off in response to various inductive mechanisms of the cell or organism; act as transcription factors by determining whether or not transcription is initiated, enhanced, or repressed; and splice transcripts as dictated in a particular cell or tissue.
  • regulatory molecules interact with short stretches of DNA scattered throughout the entire genome, most gene expression is regulated near the site at which transcription starts or within the open reading frame of the gene being expressed.
  • the regulated stretches of the DNA can be simple and interact with only a single protein, or they can require several proteins acting as part of a complex in order to regulate gene expression.
  • the double helix structure and repeated sequences of DNA create external features which can be recognized by the regulatory molecules. These external features are hydrogen bond donor and acceptor groups, hydrophobic patches, major and minor grooves, and regular, repeated stretches of sequence which cause distinct bends in the helix. Such features provide recognition sites for the binding of regulatory proteins. Typically, these recognition sites are less than 20 nucleotides in length although multiple sites may be adjacent to each other, and each may exert control over a single gene. Hundreds of these DNA sequences have been identified, and each is recognized by a different protein or complex of proteins which carry out gene regulation.
  • the regulatory protein molecules or complexes recognize and bind to specific nucleotide sequences of upstream (5′) nontranslated regions, which precede the first translated exon of the open reading frame (ORF); of intron junctions, which occur between the many exons of the OR; and of downstream (3′) untranslated regions, which follow the ORF.
  • the regulatory molecule surface features are extensively complementary to the surface features of the double helix. Even though each individual contact between the protein(s) and helix may be relatively weak (hydrogen bonds, ionic bonds, and/or hydrophobic interactions) and the 20 or more contacts occurring between the protein and DNA result in a highly specific and very strong interaction.
  • Many of the regulatory molecules incorporate one of a set of DNA-binding structural motifs, each of which contains either ⁇ helices or ⁇ sheets and binds to the major groove of DNA.
  • Seven of the structural motifs common to regulatory molecules are helix-turn-helix, homeodomains, zinc finger, steroid receptor, ⁇ sheets, leucine zipper, and helix-loop-helix.
  • the helix-turn-helix motif is constructed from two ⁇ helices connected by a short chain of amino acids, which constitutes the “turn”.
  • the two helices interact with each other to form a fixed angle.
  • the more carboxy-terminal helix is called the recognition helix because it fits into the major groove of the DNA.
  • the amino acid side chains of the helix recognize the specific DNA sequence to which the protein binds.
  • the H remaining structure varies a great deal among the regulatory proteins incorporating this motif.
  • the helix-turn-helix configuration is not stable without the rest of the protein and will not bind to DNA without other peptide regions providing stability. Other peptide regions also interact with the DNA, increasing the number of unique sequences a helix-turn-helix can recognize.
  • sequence-specific DNA binding proteins actually bind as symmetric dimers to DNA sequences that are composed of two very similar half-sites, also arranged symmetrically. This configuration allows each protein monomer to interact in the same way with the DNA recognition site and doubles the number of contacts with the DNA. This doubling of contacts greatly increases the binding affinity while only doubling the free energy of the interaction. Helix-turn-helix motifs always bind to DNA that is in the B-DNA form.
  • the homeodomain motif is found in a special group of helix-turn-helix proteins that are encoded by homeotic selector genes, so called because the proteins encoded by these genes control developmental switches. For example, mutations in these genes cause one body part to be converted into another in the fruit fly, Drosophila. These genes have been found in every eukaryotic organism studied.
  • the helix-turn-helix region of different homeodomains is always surrounded by the same structure, but not necessarily the same sequence, and the motif is always presented to DNA the same way. This helix-turn-helix configuration is stable by itself and, when isolated, can still bind to DNA.
  • helices in homeodomains are generally longer than the helices in most HLH regulatory proteins. Portions of the motif which interact most directly with DNA differ among these two families. Detailed examples of DNA-protein binding are described in Pabo and Sauer (1992; Ann Rev Biochem 61:1053-95).
  • a third motif incorporates zinc molecules into the crucial portion of the protein.
  • These proteins are most often referred to as having zinc fingers, although their structure can be one of several types. Proteins in this family often contain tandem repeats of the 30-residue zinc finger motif, including the sequence patterns Cys-X 2 or 4-Cys-X 12 -His-X 3- -His.
  • Each of these regulatory proteins has an a helix and an antiparallel ⁇ sheet. Two histidines in the ⁇ helix and 2 cysteines near the turn in the ⁇ sheet interact with the zinc ion which holds the ⁇ helix and the ⁇ sheet together.
  • the steroid receptors are a family of intracellular proteins that include receptors for steroids, retinoids, vitamin D, thyroid hormones, and other important compounds.
  • the DNA binding domain of these proteins contains about 70 residues, eight of which are conserved cysteines.
  • the steroid receptor motif forms a structure in which two a helices are packed perpendicularly to each other, forming more of a globular shape than a finger. Each helix has a zinc ion which holds a peptide loop against the N-terminal end of the helix. The first helix fits into the major groove of DNA, and side chains make contacts with edges of the DNA base pairs.
  • the steroid receptor proteins like the helix-turn-helix proteins, form dimers that bind the DNA.
  • the second helix of each monomer contacts the phosphate groups of the DNA backbone and also provides the dimerization interface.
  • multiple choices can exist for heterodimerization which produces another mechanism for fine-tuning the regulation of numerous genes.
  • Another family of regulatory protein molecules uses a motif consisting of a two-stranded antiparallel ⁇ sheet to recognize the major groove of DNA.
  • the exact DNA sequence recognized by the motif depends on the amino acid sequence in the ⁇ sheet from which the amino acid side chains extend and contact the DNA.
  • the regulatory proteins form tetramers when binding DNA.
  • the leucine zipper motif commonly forms dimers and has a 30-40 residue motif in which two ⁇ helices (one from each monomer) are joined to form a short coiled-coil.
  • the helices are held together by interactions among hydrophobic amino acid side chains (often on heptad-repeated leucines) that extend from one side of each helix. Beyond this, the helices separate, and each basic region contacts the major groove of DNA. Proteins with the leucine zipper motif can also form either homodimers or heterodimers, thus extending the specific combinations available to activate or repress expression.
  • Yet another motif is the helix-loop-helix, which consists of a short ⁇ helix connected by a loop to a longer ⁇ helix.
  • the loop is flexible and allows the two helices to fold back against each other.
  • the ⁇ helices bind both to DNA and to the HLH structure of another protein.
  • the second protein can be the same (producing homodimers) or different (producing heterodimers).
  • Some HLH monomers lack sufficient ⁇ helix to bind DNA, but they can still form heterodimers which can serve to inactivate specific regulatory proteins.
  • regulatory proteins have been identified to date, and more are being characterized in a wide variety of organisms. Most regulatory proteins have at least one of the common structural motifs for making contact with DNA, but several regulatory proteins, such as the p53 tumor suppressor gene, do not share their structure with other known regulatory proteins. Variations on the known motifs and new motifs have been and are currently being characterized (Faisst and Meyer (1992) Nucl Acids Res 20:3-26).
  • the invention features purified proteins, human regulatory molecules, collectively referred to as HRM and individually referred to as HRM-1 through HRM-49.
  • the purified protein comprises an amino acid sequence selected from SEQ ID NO:1 through SEQ ID NO:49 and portions thereof..
  • the invention provides isolated polynucleotides encoding HRM and complements of the encoding polynucleotides.
  • the polynucleotide comprises a nucleic acid sequence selected from SEQ ID NOs:50-98 and complements thereof.
  • the invention also provides a polynucleotide, or a complement or a fragment thereof, which is used as a probe to hybridize to any one of the polynucleotides of SEQ ID NOs:50-98.
  • the invention further provides a composition comprising the isolated and purified polynucleotides of SEQ ID NOs:50-98.
  • the invention provides a composition comprising a polynucleotide selected from SEQ ID NOs:50-98 and complements and fragments thereof and a reporter molecule or stabilizing moiety.
  • the invention still further provides a method for detecting expression of a polynucleotide which encodes a human regulatory molecule in a sample, the method comprising hybridizing the complement of a polynucleotide encoding HRM to nucleic acids of the sample under conditions to form a hybridization complex; and detecting hybridization complex formation, wherein complex formation indicates the expression of the polynucleotide encoding the human regulatory molecule in the sample.
  • the complement of the polynucleotide encoding HRM is immobilized on a substrate.
  • the substrate is a microarray.
  • the invention provides a vector containing at least a fragment of any one of the polynucleotides selected from SEQ ID NOs:50-98.
  • the vector is contained within a host cell.
  • the invention also provides a method for producing a protein or a portion thereof, the method comprising culturing a host cell containing a vector containing at least a fragment of a polynucleotide encoding an HRM under conditions for the expression of the protein; and recovering the protein from the host cell culture.
  • the invention further provides a composition comprising a purified HRM and a labeling moiety or a pharmaceutical carrier.
  • the invention still further provides a method for using an HRM to screen a plurality of molecules in order to obtain a ligand which specifically binds the HRM, the method comprising combining the protein with the molecules under conditions which allow specific binding, recovering the bound protein, separating the protein, thereby obtaining the ligand.
  • the molecules are selected from libraries of agonists, antibodies, antagonists, drugs, inhibitors, peptides, proteins, and pharmaceutical agents.
  • the invention still further provides a method for using a protein to produce and purify an antibody, the method comprising immunizing a animal with an HRM under conditions to elicit an antibody response; isolating animal antibodies; attaching the protein to a substrate; contacting the substrate with sera containing antibodies under conditions to allow specific binding to the HRM; dissociating the antibodies from the HRM, thereby obtaining purified antibodies.
  • the invention provides a purified antibody which specifically binds an HRM.
  • the invention also provides a method for using an antibody to detect protein expression in a sample, the method comprising combining the antibody specifically binding HRM with a sample under conditions to form antibody:protein complexes and detecting complex formation, wherein detection indicates expression of the protein in the sample.
  • expression of the HRM is diagnostic of cancer. In another aspect, expression is diagnostic of immune response.
  • the invention also provides a method for diagnosing a disease associated with gene expression in a sample containing nucleic acids, the method comprising hybridizing a polynucleotide to nucleic acids of the sample under conditions to form a hybridization complex, comparing hybridization complex formation to standards, thereby diagnosing the disease.
  • the disease is selected from a disorder characterized by cell proliferation such as a cancer, an developmental disorder, or an immune response.
  • the invention provides a method for treating a cancer comprising administering to a subject in need of such treatment a composition containing purified HRM.
  • the invention also provides a method for treating a cancer comprising administering to a subject in need of such treatment an antagonist which specifically binds HRM.
  • the invention further provides a method for treating an immune response associated with the increased expression or activity of HRM comprising administering to a subject in need of such treatment an antagonist which specifically binds HRM.
  • the invention still further provides a method for stimulating cell proliferation comprising administering purified HRM to a cell.
  • Antist refers to a molecule which specifically binds to and modulates the activity of HRM.
  • an “allele” is an alternative form of the polynucleotide or gene encoding HRM. Alleles result from at least one mutation in the nucleic acid sequence and may result in the expression of altered mRNAs or proteins whose structure or function may or may not be altered. Any given gene may have none, one, or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to additions, deletions, 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. Similarly a polynucleotide may be altered to produce deliberate amino acid substitutions.
  • substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological or immunological activity of HRM is retained.
  • negatively charged residues include aspartic acid and glutamic acid
  • positively charged residues include lysine and arginine
  • residues with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, and valine, glycine and alanine, asparagine and glutamine, serine and threonine, and phenylalanine and tyrosine.
  • Antagonist refers to a molecule which, when bound to HRM, decreases the amount or the duration of the biological or immunological activity of HRM. Antagonists may include proteins, nucleic acids, carbohydrates, fats or any other molecules which decrease the effect of HRM.
  • Antibody refers to intact molecules, or fragments thereof such as Fa, F(ab′) 2 , and Fv, which are capable of binding the antigenic determinant of an HRM.
  • Bioly active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
  • immunologically active refers to the capability of the natural, recombinant, or synthetic protein or peptide to induce a specific immune response in animals or cells and to bind with specific antibodies.
  • “Complementary” refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing.
  • the sequence “A-G-T” binds to the complementary sequence “T-C-A”.
  • the degree of complementarity between nucleic acids has significant effects on the efficiency and strength of hybridization. This is important in amplification reactions and in the design and use of peptide nucleic acid molecules.
  • composition refers to a combination comprising a plurality of polynucleotides or a specific polynucleotide or protein and at least one other molecule.
  • Such other molecules may include reporter molecules, labeling moieties, pharmaceutical carriers, carbohydrates, and the like.
  • Consensus refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, has been extended using XL-PCR kit (Applied Biosystems (ABI), Foster City Calif.) in the 5′ and/or the 3′ direction and resequenced, or has been assembled to full length from overlapping shorter fragments using a computer program for fragment assembly such as that described in U.S. Ser. No. 09/276,534, filed 25 Mar. 1999.
  • “Derivative” refers to the chemical modification of a polynucleotide or protein. Such modifications may include replacement of hydrogen by an alkyl, acyl, or amino group.
  • a nucleic acid derivative may encode a protein which retains the biological or immunological function of the natural molecule.
  • a derivative protein is one which is modified by glycosylation, pegylation, or any similar process but still retains the biological or immunological function of the native protein.
  • “Differential expression” refers to an increased, upregulated or present, or decreased, downregulated or absent, gene expression as detected by presence, absence or at least about two-fold changes in the amount of transcribed messenger RNA or translated protein in a sample.
  • disorder refers to a condition, disease or syndrome in which a polynucleotide or a protein of the invention is differentially expressed. Such a disorder includes cancers or immune responses as they are set forth below.
  • HRM refers to any one or all of the human proteins, HRMs 1-49, as it was obtained from any species including bovine, ovine, porcine, murine, equine, and preferably human, or from any source whether natural, synthetic, semi-synthetic, or recombinant.
  • Hybridization complex refers to a complex formed between two nucleic acids by the formation of hydrogen bonds between complementary base pairs; these hydrogen bonds form in an antiparallel configuration and may be further stabilized by base stacking interactions.
  • a hybridization complex may be formed in solution or between one nucleic acid present in solution and another nucleic acid immobilized on a substrate.
  • isolated refers to a polynucleotide that is removed from its natural environment or separated from other components with which it is naturally associated.
  • Ligand refers to any agent, molecule, or compound which will bind specifically to a polynucleotide or to a protein. Such ligands stabilize or modulate the activity of polynucleotides or proteins and may be composed of inorganic and/or organic substances including minerals, cofactors, nucleic acids, proteins, carbohydrates, fats, and lipids.
  • “Microarray” refers to an arrangement of distinct polynucleotides on a substrate
  • “Oligonucleotide” refers to a nucleic acid sequence about 6 nucleotides to about 60 nucleotides in length which may be used in amplification or hybridization assays. Equivalent terms include “amplimers”,“primers”, “oligomers”, and “probes”, as these are commonly defined in the art.
  • “Peptide nucleic acid” refers to an anti-gene agent which comprises an oligonucleotide of at least five nucleotides in length linked to a peptide backbone of amino acid residues which ends in a terminal lysine which confers solubility to the molecule.
  • Polynucleotide refers to nucleic acid molecule having a nucleic acid sequence and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded and represent the sense or antisense strand.
  • “Fragment” refers to a nucleic acid sequence which is more than about 60 nucleotides in length.
  • “Portion” refers to a fragment of an HRM which ranges in size from five amino acid residues to the entire amino acid sequence minus one amino acid.
  • Protein refers to an oligopeptide, peptide, or polypeptide having an amino acid sequence whether naturally occurring or synthetic molecules. Portions of HRM are preferably about 5 to about 15 amino acids in length and retain the biological or the immunological activity of the HRM.
  • “Purified” refers to a peptide or protein that is removed from its natural environment, isolated or separated from other components with which it is naturally associated.
  • Reporter molecules include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • sample is used in its broadest sense and may comprise a bodily fluid, extract from a cell, chromosome, organelle, or membrane isolated from a cell, a cell, genomic DNA, RNA, or cDNA (in solution or bound to a solid support), a tissue, a tissue print, and the like.
  • Specific binding refers to that interaction between a polynucleotide or protein of the invention and any ligand which specifically binds to it and which is selected from a DNA or an RNA molecule, a peptide nucleic acid, a peptide, a protein, an agonist, an antibody, an antagonist, an inhibitor, a mimetic, a pharmaceutical agent, a drug, a transcription factor, or an artificial chromosome construction.
  • the interaction is dependent upon the presence of a particular sequence or three dimensional structure recognized by the binding molecule.
  • Substrate refers to any rigid or semi-rigid support to which polynucleotides or proteins are bound and includes membranes, filters, chips, slides, wafers, fibers, magnetic or nomnmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores.
  • “Variant” refers to molecules that are recognized variations of a polynucleotide or a protein encoded by the polynucleotide. Splice variants may be determined by BLAST score, wherein the score is at least 100, and most preferably at least 400. Allelic variants have a high percent identity to the polynucleotides and may differ by about three bases per hundred bases. “Single nucleotide polymorphism” (SNP) refers to a change in a single base as a result of a substitution, insertion or deletion. The change may be conservative (purine for purine) or non-conservative (purine to pyrimidine) and may or may not result in a change in an encoded amino acid or its secondary, tertiary, or quaternary structure.
  • SNP single nucleotide polymorphism
  • the invention is based on the discovery of human regulatory molecules (HRM) and the polynucleotides encoding HRM, and on the use of these compositions for the diagnosis and treatment of diseases associated with cell proliferation.
  • HRM human regulatory molecules
  • Table 1 shows the protein and polynucleotide identification numbers, protein abbreviation, Incyte Clone number, cDNA library, and the closest NCBI homolog and NCBI sequence identifier for each of the human regulatory molecules.
  • HRM-1 (SEQ ID NO:1) was identified in Incyte Clone 133 from the U937NOT01 CDNA library using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:50 was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 133 (U937NOT01), 013508 (THP1PLB01), 210174 (SPLNNOT02), 1655863 (PROSTUT08), 1725724 (PROSNOT14), 1858205 (PROSNOT18), and 2646014 (OVARTUT05).
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO: 1.
  • HRM-1 is 151 amino acids in length and has four potential phosphorylation sites at T2, S14, S69, and T111.
  • HRM-1 has sequence homology with human KIAA0105 (g285947) and is found in cDNA libraries which have proliferating cells and are associated with cancer or immune response.
  • HRM-2 (SEQ ID NO:2) was identified in Incyte Clone 1762 from the U937NOT01 cDNA library using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:51 was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 1762 (U937NOT01), 1254927 (LUNGFET03), and 2070865 (ISLTNOT01).
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:2.
  • HRM-2 is 185 amino acids in length and has a potential N glycosylation site at N108; eight potential phosphorylation sites at T22, S26, T27, S31, T51, T70, and T135; a leucine zipper motif at L 136 KDVVWGLNSLFTDLLNFDDPL; and a ubiquitin conjugation motif at W 105 HPNITETGEICLSL.
  • HRM-2 has sequence homology with a gene from Ascaris suum (g1518121) and is found in cDNA libraries which have secretory or proliferating cells and are associated with development.
  • HRM-3 (SEQ ID NO:3) was identified in Incyte Clone 1847 from the U937NOT01 cDNA library using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:52 was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 274 (U937NOT01), 1847 (U937NOT01), 262233 (HNT2AGTO1), 972977 (MUSCNOT02), and 1859611(PROSNOT18).
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:3.
  • HRM-3 is 59 amino acids in length and has four potential N glycosylation sites at N147, N352, N410, and N421, and 17 potential phosphorylation sites at S13, T21, S43, S89, S131, S207, T243, S278, T286, S335, S337, S350, S354, S369, S380, S412, and S542.
  • HRM-3 has sequence homology with a saccharomyces cerevisiae protein (g130221 1) and is found in cDNA libraries which have proliferating or immortalized cells.
  • HRM-4 (SEQ ID NO:4) was identified in Incyte Clone 9337 from the HMC1NOT01 cDNA library using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:53 was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 9337 (HMC1NOT01), 670279 (CRBLNOT01), 717305 (PROSTUT01), 968249 (BRSTNOT05), and 1546506 (PROSTUT04).
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:4.
  • HRM-4 is 338 amino acids in length and has a potential N glycosylation site at N327, 11 potential phosphorylation sites at T15, S36, S42, S50, T51, S73, S144, S176, T256, S140, and T329; and five zinc finger motifs at C 192 RC 194 SECGKI FRNPRYFSVHKKIH, C 222 QDCGKGFVQSSSLTQHQRVH, C 250 OQECGRTFNDRSAISQHLRTH, C 278 QDCGKAFRQSSHLIRHQRTH, and C 306 NKCGKAFTQSSHLIGHQRTH.
  • HRM-4 has sequence homology with a human zinc finger protein (g1613852) and is found in cDNA libraries which have proliferating, cancerous, or secretory cells.
  • HRM-5 (SEQ ID NO:5) was identified in Incyte Clone 9476 from the HMCINOT01 cDNA library using a computer search for amino acid sequence alignments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:5.
  • HRM-5 is 456 amino acids in length and has a potential N glycosylation site at N385; 14 potential phosphorylation sites at T9, T12, S58, T74, T163, T139, S175, T211, T239, T272, S331, T367, T420, and S443, and an ATP/GTP binding motif at G 70 PPGTGKT77.
  • HRM-5 has sequence homology with a S. cerevisiae protein (g755784) and is found in cDNA libraries which have dividing, cancerous or immortalized cells and are associated with immune response.
  • HRM-6 (SEQ ID NO:6) was identified in Incyte Clone 10370 from the THP1PLB01 cDNA library using a computer search for amino acid sequence alignments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:6.
  • HRM-6 is 210 amino acids in length and has one potential N-glycosylation site at N11 and nine potential phosphorylation sites at T13, T21, T46, T124, S125, S132, T143, T167, and T191.
  • HRM-6 has sequence homology with a putative p64 CLCP human protein (g895845) and is found in cDNA libraries which have dividing, cancerous or immortalized cells and are associated with immune response.
  • HRM-7 (SEQ ID NO:7) was identified in Incyte Clone 30137 from the THP1PLB01 cDNA library using a computer search for amino acid sequence alignments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:7.
  • HRM-7 is 255 amino acids in length and has one potential N glycosylation site at N86 and 12 potential phosphorylation sites at T9, T28, S32, S61, S94, S142, S156, S160, T169, S118, S220, and S236.
  • HRM-7 has sequence homology with human clone 23733 (g1710241) and is found in cDNA libraries which have dividing, cancerous or immortalized cells and are associated with immune response.
  • HRM-8 (SEQ ID NO:8) was identified in Incyte Clone 77180 from the SYNORAB01cDNA library using a computer search for amino acid sequence alignments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:8.
  • HRM-8 is 188 amino acids in length and has one potential amidation site, Q170GKR; two potential N glycosylation sites at N60 and N68; and four potential phosphorylation sites at S70, T164, T166, and S183.
  • HRM-8 has sequence homology with a S. cerevisiae protein (g5372) and is found in cDNA libraries which have dividing, cancerous or immortalized cells and are associated with immune response.
  • HRM-9 (SEQ ID NO:9) was identified in Incyte Clone 98974 from the PITUNOR01 cDNA library using a computer search for amino acid sequence alignments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:9.
  • HRM-9 is 531 amino acids in length and has one potential N glycosylation site at N480; 37 potential phosphorylation sites at S19, T22, S38, T64, T76, T91, Si117, Si118, S158, T164, T177, T182, T200, T267, Y281, Y311, Y322, S333, S394, S402, S404, S409, S414, S416, S418, S429 S434, S439, S440, S456, S460, S466, S478, S505, S510, S524, S528, and one potential glycosaminoglycan motif at S434GSG.
  • HRM-10 (SEQ ID NO: 10) was identified in Incyte Clone 118160 from the MUSCNOT01 cDNA library using a computer search for amino acid sequence alignments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:10.
  • HRM-10 is 348 amino acids in length and has two potential N glycosylation sites at N150 and N317; 17 potential phosphorylation sites at T23, T45, S60, T126, S130, S140, S145, S151, S154, S158, S 186 , Y208, Y234, S217, T271, T303, and S327, and a transcription factor signature at C 310 SKCKKKNCTYNQVQTRSA DEPMTTFVLCNEC.
  • HRM-10 has sequence homology with a Mus musculus protein (g220594) and is found in cDNA libraries which have secretory or immune associations.
  • HRM-11 (SEQ ID NO:11) was identified in Incyte Clone 140516 from the TLYMNOR01 cDNA library using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:60 was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 140516 (TLYMNOR01), 143729 (TLYMNOR01), 1346014 (PROSNOT11), and 2074866 (ISLTNOT01).
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:11.
  • HRM-11 is 393 amino acids in length and has 14 potential phosphorylation sites at S22, T33, S41, S69, T156, Y157, S166, S199, T242, T308, T324, S350, T359, S378.
  • HRM-11 has sequence homology with a C. elegans protein (g1086723) and is found in cDNA libraries which have proliferating, secretory or immune cells.
  • HRM-12 (SEQ ID NO: 12) was identified in Incyte Clone 207452 from the SPLNNOT02 cDNA library using a computer search for amino acid sequence alignments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:12.
  • HRM-12 is 320 amino acids in length and one potential amidation site at E 210 GKK; two potential N glycosylation sites atN12 and N314; seven potential phosphorylation sites at S34, S51, S56, Slll, T157, S198, and S318; one potential glycosaminoglycan motif, S224GAG; one immunoglobulin major histocompatibility motif, F 305 FCNVFH; and two mitochondrial carrier protein signatures, P 35 FDVIKIRF and P 138 VDVLRTRF.
  • HRM-12 has sequence homology with a S. cerevisiae protein (gl31 4 086) and is found in cDNA libraries which have secretory and proliferating cells.
  • HRM-13 (SEQ ID NO: 13) was identified in Incyte Clone 208836 from the SPLNNOT02 cDNA library using a computer search for amino acid sequence alignments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:13.
  • HRM-13 is 343 amino acids in length and has one potential N glycosylation site at N172; 17 potential phosphorylation sites at S45, S46, T62, S73, S84, S85, S102, S105, T124, S137, Y153, T192, S216, Y226, Y241, S253 and T293; and a zinc finger motif at C 277 RHYFCESCA.
  • HRM-13 has sequence homology with a S. cerevisiae protein (g662126) and is found in cDNA libraries which have proliferating cells and are associated with immune response.
  • HRM-14 (SEQ ID NO:14) was identified in Incyte Clone 569710 from the MMLR3DT01 cDNA library using a computer search for amino acid sequence alignments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:14.
  • HRM-14 is 368 amino acids in length and has 10 potential phosphorylation sites at S5, T16, T125, S132, S142, S157, S167, S185, S208, and S246; and four zinc finger motifs at C 253 DECGKHFSQGSALILHQRIH, C 281 ,VECGKAFSRSSILVQH QRVH, C 309 LECGKAFSQNSGLINHQRIH, and C 337 VQCGKSYSQSSNLFRHQRRH.
  • HRM-14 has sequence homology with a human zinc finger protein (gl698719) and is found in cDNA libraries which are associated with immune response.
  • HRM-15 (SEQ ID NO:15) was identified in Incyte Clone 606742 from the BRSTTUT01 cDNA library using a computer search for amino acid sequence alignments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:15.
  • HRM-15 is 158 amino acids in length and has two potential myristylation sites, G92GFHGQ and G96QMHSR, and one potential PKC phosphosphorylation site, S40.
  • HRM-15 has sequence homology with human clone 23679 (g1710201) and is found in cDNA libraries with proliferating, secretory and/or cancerous cells.
  • HRM-16 (SEQ ID NO: 16) was identified in Incyte Clone 611135 from the COLNNOT01 cDNA library using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:65 was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 611135 (COLNNOT01), 659029 (BRAINOT03), and 1861691 (PROSNOT19).
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:16.
  • HRM-16 is 334 amino acids in length and has 11 potential phosphorylation sites at S17, T29, T128, S133, S162, S176, S263, T257, S263, S277, and S294.
  • HRM-16 has sequence homology with a C. elegans protein (g506882) and is found in cDNA libraries with secretory cells.
  • HRM-17 (SEQ ID NO: 17) was identified in Incyte Clone 641127 from the BRSTNOT03 cDNA library using a computer search for amino acid sequence alignments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO: 17.
  • HRM-17 is 488 amino acids in length and has one N glycosylation site at N215; 11 potential phosphorylation sites at S70, S78, S92, T102, S111, T190, Y235, S303, S329, S415, and T471; and eight zinc finger motifs at C 237 EQCGKGFTRSSSLLIHQAVH, C 265 DKCGKGFTRSSSLLIHHAVH, C 293 DKCGKGFSQSSKLHIHQRVH, C 321 ,EECGMSFS QRSNLHIHQRVH, C 349 GECGKGFSQSSNLHIHRCIH, C 377 YECGKGFSQSSDLRIHLRVH, C 405 GKCGKGFSQSSKLLIHQRVH, and C 433 SKCGKGFSQSSNLHIHQRVH.
  • HRM-17 has sequence homology with a human
  • HRM-18 (SEQ ID NO:18) was identified in Incyte Clone 691768 from the LUNGTUT02 cDNA library using a computer search for amino acid sequence alignments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:18.
  • HRM-18 is 255 amino acids in length and has one potential N glycosylation site at N102 and 13 potential phosphorylation sites at S21, T90, T109, S111, T124, S134, S139, T141, S158, S172, S181, S187, and T206.
  • HRM-18 has sequence homology with a M. musculus protein (g309183) and is found in cDNA libraries with proliferating or cancerous cells.
  • HRM-19 (SEQ ID NO:19) was identified in Incyte Clone 724157 from the SYNOOAT01 cDNA library using a computer search for amino acid sequence alignments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO: 19.
  • HRM-19 is 351 amino acids in length and has eight potential phosphorylation sites at T30, S41, S53, T135, S172, S187, T273, and S331; one potential glycosaminoglycan site, S 18 GTG; and one potenti mitochondrial carrier motif, P 13 ,LDVVKVRL.
  • HRM-19 has sequence homology with C. elegans C16C10 (g577542) and is found in cDNA libraries associated with cell proliferation, cancer and immune response.
  • HRM-20 (SEQ ID NO:20) was identified in Incyte Clone 864683 from the BRAITUT03 cDNA library using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:69 was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 486297 (HNT2RATO1), 864683 (BRAITUT03), 1314465 (BLADTUT02), 1610776 (COLNTUT06), 1856771 (PROSNOT18), 1866081 (PROSNOT19), 1932221 (COLNNOT16), and 2125225 (BRSTNOT07).
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:20.
  • HRM-20 is 535 amino acids in length and has three potential N glycosylation sites at N202, N252, and N523; and 17 potential phosphorylation sites at S2, S12, S42, S49, S102, S157, T165, T171, T232, T255, T317, S332, S428, T441, S453, S500, and S509.
  • HRM-20 has sequence homology with a C. elegans protein (g1418563) and is found in cDNA libraries associated with cell proliferation, cancer and immune response.
  • HRM-21 SEQ ID NO:21
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:21.
  • HRM-21 is 201 amino acids in length and has one potential N glycosylation site at N82; five potential phosphorylation sites at T70, S83, S98, S154, and Ti 87; and one tyrosine phosphatase motif at V 130 HCKAGRSRSATM.
  • HRM-21 has sequence homology with a C. elegans protein (g1657672) and is found in cDNA libraries associated with immune response.
  • HRM-22 SEQ ID NO:22
  • Incyte Clone 1404643 was identified in Incyte Clone 1404643 from the LATRTUT02 cDNA library using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:71 was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 878243 (LUNGAST01), 1404643 (LATRTUT02), 1508343 (LUNGNOT14) and 2585156 (BRAITUT22).
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:22.
  • HRM-22 is 239 amino acids in length and has four potential phosphorylation sites at S5, S89, S133, and T211.
  • HRM-22 has sequence homology with a C. elegans protein (g459002) and is found in cDNA libraries associated with cell proliferation, cancer and immune response.
  • HRM-23 (SEQ ID NO:23)
  • Incyte Clone 1561587 was identified in Incyte Clone 1561587 from the SPLNNOT04 cDNA library using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:72 was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 522573 (MMLR2DTO1), 773822 (COLNNOT05), 1304839 (PLACNOT02), 1381253 (BRAITUT08), 1452511 (PENITUT01), 1539060 (SINTIUT01), 1561587 (SPLNNOT04), and 2416572 (HNT3AZTO1).
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:23.
  • HRM-23 is 244 amino acids in length and has five potential phosphorylation sites at T40, S75, T84, T89, and S194.
  • HRM-23 has sequence homology with a C. elegans protein (g868266) and is found in cDNA libraries associated with cell proliferation, cancer and immune response.
  • HRM-24 (SEQ ID NO:24) was identified in Incete Clone 1568361 from the UTRSNOT05 cDNA library using a computer search for amino acid sequence.
  • a consensus sequence, SEQ ID NO;73 was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 927874 (BRAINOT04), 1255220 (MENITUT03), 1242340 (LUNGNOT03), 13495 (LATRTU02), 1381263 (BRAITUT08), 1500028 (SINTBST01), 1568361 (UTRSNOT05), 1653237 (PROSTUT08), 1975340 (PANCTUT02), and 3274608 (PROSBPT06).
  • the invention encompasses a protein compraising the amino acid sequence of SEQ ID NO:24, HRM-24 is 431 amino acids in length and has five potential N glycosylation sites at N75, N95 N171, N202, and N298; eight potential phosphorylation sites at S2, S3, T11, T13, S17, Y316, T375, and T415, and a leucine zipper motif, L 96 SAFNNILSNLGYILLGLLFLL. HRM-24 has sequence homology with human mucin (g1834503) and is found cDNA libraries proliferating, cancerus or inflamed cells.
  • HRM-25 (SEQ ID NO;25) was identified in Incyte Clone 1572888 from the LNODNOT03 cDNA library using a computer search for amino acid sequence aligments.
  • a consensus sequence SEQ ID NO:74 was derived from the extended and overlapping nucleic acid sequence: Incyte Clones 1438142 (PANCNT08), 1572888 (LNODNOT03), and 1665075 (BRSTNOT09).
  • the invention encompases a protein compraising the amino acid sequence of SEQ ID NO:25.
  • HRM-25 is 376 amino acids in length and has one N glycosylation site five potential phosphorylation sites at S111, T150, S151, T159, and, S196.
  • HRM-25 has sequence homology with S. cerevisiae YER156c (g603396) and is found in cDNA libraries with secretory cells.
  • HRM-26 (SEQ ID NO:26) was identifie in Incyte Clone 1573677 from the LONDNOT03 cDNA library using a computer search for amino acid sequence aligments.
  • a concensus sequence, SEQ ID NO:75 was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 040360 (TBLYNOT01), 065573 (PLACNOB01), 228382 (PANCNOT01), 1457788 (COLNFET02), 1573677 (LNODNOT03), and 1854560 (HNT3AZT01)
  • the invention encompasses a protein compraising the amino acid sequence of SEQ ID NO:26 is 340 amino acids in length and has one potential N glycosylation site at N213 and 13 potential phosphorylation site at T10, S22, T53, T56, S160, S168, S170, S177, S201, S226 S297, S303, and T329.
  • HRM-26 has sequence homology with S. cerevisiae D9481.16 (g849195) and its found in cNDA libraries associated with secretion, immune response, and cancer.
  • HRM-27 (SEQ ID NO:27) was identified in Icyte Clone 1574624 from the LNODNOT03 cDNA library using a coomputer search for amino acid sequence aligments.
  • a concesnsus sequence, SEQ ID NO:76 was derived from the extended and overlapping nucleic acid sequence: Incyte Clones 90012 (HYPONOB01), 888491 (STOMTUT01), and 1574624 (LNODNOT03).
  • the invention encompasses a protein comprising the amino acids sequence of SEQ ID NO:27.
  • HRM-27 is 174 amino acids in length and has one N glycosylation site at N51 and five potential phosphorylation sites at S111, T150, S151, T159, and T196.
  • HRM-27 has sequence homology with a C. elegants protein (g1067025) and is found in cDNA libraries associated with secretion, immune responce and ancer.
  • HRM-28 (SEQ ID NO:28) was identified in Incyte Clones 1577239 from the LNODNOT03 cDNA library using a computer search for amino acid sequence aligments.
  • the invention encompasses a protein comprising the amino acids sequence of SEQ ID NO:28, HRM-28 is 179 amino acids in length and has one potential N glycosylation site at N60 and five potential phosphorylation site at Y61, S62, Y104, T136, and Y142.
  • HRM-28 has sequence homology with a S. cerevisiae protein (g728657) and is found in cDNA libraries associated with sevretion and immune response.
  • HRM-29 (SEQ ID NO:29) was identified in Incyte Clone 1598203 from the BLADNOT-03 cDNA library using a computer search for amino acid sequence aligments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO;29.
  • HRM-29 is 205 amino acids in length and has one potential N glycocylation site at N117 and five potential phosphorylation sites at T68, T118, S137, S140, and S159.
  • HRM-29 has sequence homology with a C. elegants protein (g1200033) and is found in cDNA libraries associated with secretion.
  • HRM-30 (SEQ ID NO:30) was identified in Incyte Clone 1600438 fron the BLADNOT03 cDNA library using a computer search for amino acid sequence aligments.
  • the invention encompasses a protein comprising the acid sequenceof SEQ ID NO:30.
  • HRM-30 is 419 amino acids in length and has one potential N glycosylation site at N161; twelve potential phosphorylation sites at T16, S57, T67, T83, S100, T107, S144, S206, T254, Y351, S412, and S414; a leucine zipper motif, l 38 NEAGDDLEAVAKFLDSGSRL; and an ATP/GTP binding motif, A 385 HVAKGKS.
  • HRM-30 has sequence homology with human KIAA0005 (g286001) and is found in cDna libraries associated with secretion and cancer.
  • HRM-31 (SEQ ID NO:31) was identified in Incyte Clone 1600518 from the BLADNOT03 cNDA library using a computer search for amino acid sequence aligment.
  • a consensus sequence, SEQ ID NO;80 was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 389679 (THYMNOT02), 1600518 (BLADNOT03), 2055734 (BEPINOT02), and 2509270 CONUTUT01).
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:31.
  • HRM-31 is 376 amino acid in length and has one potential N glycosylation site at N161 and 14 potential phosphorylation sites at T30, S65, S75, S95, S106, T134, S159, S224, T228, T250, T292, S299, T303, and S323 and a glycosaminoglycan motif, S14 GPG.
  • HRM-31 has sequence homology with a C. elegants protein (g790405) and is found in cNDA libraries associated with immune response, secretion and cancer.
  • HRM-32 (SEQ ID NO:32) was identified in Incyte Clone 1602473 from the BLADNOT03 cDNA library using a computer search for amino acid sequence aligments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:32.
  • HRM-32 is 237 amino acids in length and has seven potential phosphorylation site at T51, T68, S92, S143, T171, S193, and S203.
  • HRM-32 has sequence homology with a Haemophilus influenzae protein (g1574570) and is found in cDNA libraries associated with immune response, and cancer.
  • HRM-33 (SEQ ID NO:33) was identified in Incyte Clone 16057220 from the LUNGNOT15 cDNA library using a computer search for amino acid sequence aligment.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:33.
  • HRM-33 is 152 amino acids in length and has four potential phosphorylation sites at S10, S23, T34, and S66; and a leucine zipper motif, L 77 AVGNYRLKEYEKALKYVRGLL.
  • HRM-33 has sequence homology with C. elegans (g155080) and is found in cDNA libraries associated with secretion and immune response.
  • HRM-34 (SEQ ID NO:34) was identified in Incyte Clone 1610501 from the COLNTUT06 cDNA library using a computer search for amino acid sequence alignments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:34.
  • HRM-34 is 179 amino acids in length and has five potential phosphorylation sites at S32, S48, T45, T50, and T52.
  • HRM-34 has sequence homology with a S. cerevisiae protein (g313741) and is found in cDNA libraries associated with cancer and immune response.
  • HRM-35 (SEQ ID NO:35) was identified in Incyte Clone 1720770 from the BLADNOT06 cDNA library using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:84 was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 681455 (UTRSNOT02), 813292 (LUNGNOT04), 1223029 (COLNTUT02), 1444186 (THYRNOT03), 1522592 (BLADTUT04), 1720770 (BLADNOT06), and 1798409 (COLNNOT27).
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:35.
  • HRM-35 is 196 amino acids in length and has an amidation motif, H 17 9GKR, and seven potential phosphorylation sites at S2, S6, S31,S84, S90, T136, and T161.
  • HRM-35 has sequence homology with a C.elegans protein (g1006641) and is found in cDNA libraries associated with secretion, immune response, and cancer.
  • HRM-36 (SEQ ID NO:36) was identified in Incyte Clone 1832295 from the BRAINON01 cDNA library using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:85 was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 060275 (LUNGNOT01), 1823989 (GBLATUT01), and 1832295 (BRAINON01).
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:36.
  • HRM-36 is 612 amino acids in length and has 12 potential N glycosylation sites at N36, N95, N139, N146, N151, N176, N188, N226, N243, N353, N371, and N482; and 16 potent at S58, S92, S112, T153, T198, T248, S308, S373, T400, T420, T428, Y438, T458, T472, S527, and S556.
  • HRM-36 has sequence homology with human enigma protein (g561 6 37) and is found in cDNA libraries associated with secretion and immune response.
  • HRM-37 (SEQ ID NO:37) was identified in Incyte Clone 1990522 from the CORPNOT02 cDNA library using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:86 was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 264363 (HNT2AGTO0), 1990522 (CORPNOT02), and 2451448 (ENDANOT01).
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:37.
  • HRM-37 is 101 amino acids in length and has a PKC phosphorylation site at S62.
  • HRM-37 has sequence homology with a S. cerevisiae protein (g558396) and is found in cDNA libraries associated with immune response.
  • HRM-38 (SEQ ID NO:38) was identified in Incyte Clone 2098087 from the BRAITUT02 cDNA library using a computer search for amino acid sequence alignments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:38.
  • HRM-38 is 132 amino acids in length and has a potential ATP/GTP binding motif at G 74 ARNLLKS.
  • HRM-38 has sequence homology with M. musculus uterine protein (g166284) and is found in cDNA libraries associated with immune response.
  • HRM-39 (SEQ ID NO:39) was identified in Incyte Clone 2112230 from the BRAITUT03 cDNA library using a computer search for amino acid sequence alignments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:39.
  • HRM-39 is 188 amino acids in length and has a potential N glycosylation site at N87 and eight potential phosphorylation sites at T10, T28, S74, S93, T121, T128, Y168, and T169.
  • HRM-40 (SEQ ID NO:40) was identified in Incyte Clone 2117050 from the BRAITUT02 cDNA library using a computer search for amino acid sequence alignments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:40.
  • HRM-40 is 86 amino acids in length and has a potential N glycosylation site at N58 and four potential phosphorylation sites at T2, S9, T26, and T27.
  • HRM-40 has sequence homology with a C. elegans protein (g687821) and is found in cDNA libraries involved in cell proliferation, secretion, cancer, and immune response.
  • HRM-41 (SEQ ID NO:41) was identified in Incyte Clone 2184712 from the SININOT01 cDNA library using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:90 was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 922736 (RATRNOT02), 1976003 (PANCTUT02), and 2184712 (SININOT01).
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:41.
  • HRM-41 is 222 amino acids in length and has a potential amidation site, K 10 GKK; a potential glycosaminoglycan site, S 2 GLG; a potential N glycosylation site, N95; and seven potential phosphorylation sites at T18, T29, T50, S84, T98, S112, and S188.
  • HRM-41 has sequence homology with a C. elegant protein (g868241) and is found in cDNA libraries involved in cell proliferation, secretion, cancer, and immune response.
  • HRM-42 (SEQ ID NO:42) was identified in Incyte Clone 2290475 from the BRAINON01 cDNA library using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:91 was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 238339 (SINTNOT02), 1657945 (URETTUT01), 1848691 (LUNGFET03), 2044604 (THPlT7T01), 2290475 (BRAINON01), and 2514944 (LIVRTUT04).
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:42.
  • HRM-42 is 300 amino acids in length and has a potential N glycosylation site, N5; seven potential phosphorylation sites at S23, S71, S132, S142, T176, T192, and S293; and a Mutt signature, G 165 MVDPGEKISATLKREFGEE.
  • HRM-42 has sequence homology with a C. elegans protein (g733605) and is found in cDNA libraries involved in cell proliferation, secretion, cancer, and immune response.
  • HRM-43 (SEQ ID NO:43) was identified in Incyte Clone 2353452 from the LUNGNOT20 cDNA library using a computer search for amino acid sequence alignments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:43.
  • HRM-43 is 112 amino acids in length and has six potential phosphorylation sites at T23, T43, S44, T79, T84, and T98.
  • HRM-43 has sequence homology with a Schizosaccharomvces pombe protein (gl507666) and is found in cDNA libraries involved in cell proliferation, secretion, cancer, and immune response.
  • HRM-44 (SEQ ID NO:44) was identified in Incyte Clone 2469611 from the THPlNOT03 cDNA library using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:93 was derived from the extended and overlapping nucleic acid sequences: Incyte clones 003088 (HMClNOT01), 1448981 (PLACNOT02), 1453563 (PENITUT01), 1824146 (GBLATUT01), 2369282 (ADRENOT07), 2469611 (THPlNOT03), and 2622587 (KERANOT02).
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:44.
  • HRM-44 is 251 amino acids in length and has a potential glycosaminoglycan site, S218GFG, and four potential phosphorylation sites at T8, S83, S212, and S226.
  • HRM-44 has sequence homology with a C. elegans protein (gl495332) and is found in cDNA libraries involved in cell proliferation, secretion, cancer, and immune response.
  • HRM-45 (SEQ ID NO:45) was identified in Incyte Clone 2515476 from the LIVRTUT04 cDNA library using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:94 was derived from the extended and overlapping nucleic acid sequences: Incyte clones 18414 (HUVELPB01), 78341 (SYNORAB01), 143277 (TLYMNOR01), 181574 (PLACNOB01), 832996 (PROSTUT04), 962753 (BRSTTUT03), 1413604 (BRAINOT12), and 2515476 (LIVRTUT04).
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:45.
  • HRM-45 is 811 amino acids in length and has three potential amidation sites at G 113 GRR, W 165 GKR, and G 790 GKK; four potential N glycosylation sites at N22, N56, N79, and N145; 24 potential phosphorylation sites at T11, S13, S30, S60, Y71, S81, S85, S86, S103, S254, S256, T377, S388, S425, S456, S487, T544, S552, S574, T659, S678, S702, S746, and S753; a potential glycosaminoglycan site, S 160 GHG; and a potential zinc finger motif at C 240 GHIFCWACI.
  • HRM-45 has sequence homology with human KIAA0262 (g1665790) and is found in cDNA libraries involved in cell proliferation, secretion, cancer, and immune response
  • HRM-46 (SEQ ID NO:46) was identified in Incyte Clone 2754573 from the THPlAZSO8 cDNA library using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:95 was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 263630 (HNT2AGTO1), 412307 (BRSTNOT01), 491644 (HNT2AGTO1), 1253094 (LUNGFET03), 2270603 (PROSNON01), 2280508 (PROSNON01), 2375670 (ISLTNOT01), 2754573 (THPlAZS08), and 3151587 (ADRENON04).
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:46.
  • HRM-46 is 352 amino acids in length and has two potential N glycosylation sites at N141 and N294, and thirteen potential phosphorylation sites at S8, T67, T106, T110, T121, S122, S169, S206, T210, S215, S256, S260, and T296.
  • HRM-46 has sequence homology with human RNA binding protein (g478990) and is found in cDNA libraries involved in cell proliferation, secretion, and immune response.
  • HRM-47 (SEQ ID NO:47) was identified in Incyte Clone 2926777 from the TLYMNOT04 cDNA library using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:96 was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 040208 (TBLYNOT01), 900242 (BRSTTUT03), 963500 (BRSTTUT03), 1996474 (BRSTTUT03), and 2926777 (TLYMNOT04).
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:47.
  • HRM-47 is 432 amino acids in length and has a potential N glycosylation site at N417 and 24 potential phosphorylation sites at T51, S73, T122, T133, S177, S206, T226, T238, S293, S300, S304, S309, T325, S333, S339, S353, S360, Y361, S384, S390, T403, T412, T419, and S425 homology with a C. elegans protein (g687823) and is found in cDNA libraries involved in cell proliferation, secretion, cancer, and immune response.
  • C. elegans protein g687823
  • HRM-48 (SEQ ID NO:48) was identified in Incyte Clone 3217567 from the TESTNOT07 cDNA library using a computer search for amino acid sequence alignments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:48.
  • HRM-48 is 180 amino acids in length and has a potential zinc finger motif, C42GHLYCWPCL, and five potential phosphorylation sites at T33, T57, S84, T148, and S160.
  • HRM-48 has sequence homology with human HLA class III region (g1841547) and is found in cDNA libraries involved in secretion and immune response.
  • HRM-49 (SEQ ID NO:49) was identified in Incyte Clone 3339274 from the SPLNNOT10cDNA library using a computer search for amino acid sequence alignments.
  • the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:49.
  • HRM-49 is 137 amino acids in length and has three potential phosphorylation sites at Ti 11, T91, and S119.
  • HRM-49 has sequence homology with a deduced human translational inhibitor (g1177434) and is found in cDNA libraries involved in secretion and immune response.
  • the invention also encompasses HRM variants which retain the biological or functional activity of HRM.
  • a preferred HRM variant is one having at least 60% amino acid sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-49.
  • the invention also encompasses polynucleotides which encode HRM. Accordingly, any nucleic acid sequence which encodes the amino acid sequence of HRM can be used to produce recombinant molecules which express HRM. In a particular embodiment, the invention encompasses a polynucleotide comprising a nucleic acid sequence selected from SEQ ID NOs:50-98 and fragment and complements thereof.
  • polynucleotides which encode HRM and its variants are preferably capable of hybridizing to the polynucleotide of the naturally occurring HRM under selected conditions of stringency, it may be advantageous to produce polynucleotides encoding HRM or its derivatives possessing a different codon usage. 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 transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • the invention also encompasses production of polynucleotides, or fragments thereof, which encode HRM and its derivatives, entirely by synthetic chemistry.
  • the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents that are well known in the art.
  • synthetic chemistry may be used to introduce mutations into a sequence encoding HRM or any fragment thereof.
  • polynucleotides that are capable of hybridizing to the nucleic acids of a sample, and in particular, the polynucleotides or the complements thereof shown in SEQ ID NOs:50-98, under various conditions of stringency as taught in Wahl and Berger (1987; Methods Enzymol 152:399-407) and Kimmel (1987; Methods Enzymol 152:507-511).
  • Methods for DNA sequencing which are well known and generally available in the art and may be used to practice any of the embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE, Taq DNA polymerase and thermostable T7 DNA polymerase (Amersham Pharmacia Biotech (APB), Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD).
  • the process is automated with machines such as the MICROLAB system (Hamilton, Reno NV), DNA ENGINE thermal cycler (MJ Research, Watertown MA), and the Catalyst preparation and 373 and 377 PRISM DNA sequencing systems (ABI).
  • machines such as the MICROLAB system (Hamilton, Reno NV), DNA ENGINE thermal cycler (MJ Research, Watertown MA), and the Catalyst preparation and 373 and 377 PRISM DNA sequencing systems (ABI).
  • the nucleic acid sequences encoding HRM may be extended utilizing a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements.
  • one method which may be employed, “restriction-site” PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar (1993) PCR Methods Applic 2:318-322).
  • genomic DNA is first amplified in the presence of primer to a linker sequence and a primer specific to the known region.
  • the amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one.
  • Products of each round of PCR are transcribed with an RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR may also be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al. (1988) Nucleic Acids Res 16:8186).
  • the primers may be designed using commercially available software such as OLIGO software (Molecular Insights, Cascade CO), or another program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72 C.
  • the method uses several restriction enzymes to generate a fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al. (1991) PCR Methods Applic 1:111-119).
  • capture PCR involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al. (1991) PCR Methods Applic 1:111-119).
  • multiple restriction enzyme digestions and ligations may also be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
  • Another method which may be used to retrieve unknown sequences is that of Parker et al. (1991; Nucleic Acids Res 19:3055-3060).
  • libraries that have been size-selected to include larger cDNAs.
  • random-primed libraries are preferable, in that they will contain more sequences which contain the 5′ regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries may be useful for extension of sequence into 5′ non-transcribed regulatory regions.
  • Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the sequence of sequencing or PCR products.
  • capillary sequencing may employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera.
  • Output/light intensity may be converted to electrical signal using software integral to the system, 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 the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
  • polynucleotides or fragments thereof which encode HRM may be used in recombinant DNA molecules to direct expression of HRM, portions or functional equivalents thereof, in host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode the same or a functionally equivalent amino acid sequence may be produced, and these sequences may be used to clone and express HRM.
  • HRM-encoding polynucleotides possessing non-naturally occurring codons it may be advantageous to produce HRM-encoding polynucleotides possessing non-naturally occurring codons.
  • codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
  • the polynucleotides of the present invention can be engineered using methods generally known in the art in order to alter HRM encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the polynucleotides.
  • site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • natural, modified, or recombinant nucleic acid sequences encoding HRM may be ligated to a heterologous sequence to encode a fusion protein.
  • a heterologous sequence For example, to screen peptide libraries for inhibitors of HRM activity, it may be useful to encode a chimeric HRM protein that can be recognized by a commercially available antibody.
  • a fusion protein may also be engineered to contain a a cleavage site located between the HRM encoding sequence and the heterologous protein sequence, so that HRM may be cleaved and purified away from the heterologous moiety.
  • sequences encoding HRM may be synthesized, in whole or in part, using chemical methods well known in the art (Caruthers et al. (1980) Nucleic Acids Symp Ser. (7) 215-223, Horn et al. (1980) Nucleic Acids Symp. Ser. (7) 225-232).
  • the protein itself may be produced using chemical methods to synthesize the amino acid sequence of HRM, or a portion thereof.
  • peptide synthesis can be performed using various solid-phase techniques (Roberge et al. (1995) Science 269:202-204) and automated synthesis may be achieved, for example, using the 43 1A Peptide synthesizer (ABI).
  • the newly synthesized peptide may be purified by preparative high performance liquid chromatography (see Creighton (1983) Proteins Structures and Molecular Principles, WH Freeman, New York N.Y.).
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra).
  • amino acid sequence of HRM, or any part thereof may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant protein.
  • the polynucleotides encoding HRM or functional equivalents may be inserted into expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • a variety of expression vector/host systems may be utilized to contain and express sequences encoding HRM. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with baculovirus expression vectors; plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) or with bacterial expression vectors (Ti or pBR322 plasmids); or animal cell systems.
  • the invention is not limited by the host cell employed.
  • control elements are those non-translated regions of the vector enhancers, promoters, 5′ and 3′ untranslated regions--which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla CA) or the pSport1 plasmid (Life Technologies) may be used. The baculovirus polyhedrin promoter may be used in insect cells.
  • Promoters or enhancers derived from the genomes of plant cells e.g., heat shock, RUBISCO; and storage protein genes
  • plant viruses e.g., viral promoters or leader sequences
  • promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding HRM, vectors based on SV40 or EBV may be used with an selectable marker.
  • a number of expression vectors may be selected depending upon the use intended for HRM. For example, when large quantities of HRM are needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be used. Such vectors include, but are not limited to, the multifunctional E.
  • coli cloning and expression vectors such as BLUESCRIPT phagemid (Stratagene), in which the sequence encoding HRM may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke and Schuster (1989) J Biol Chem 264:5503-5509); and the like.
  • pGEX vectors may also be used to express foreign proteins as fusion proteins with glutathione S-transferase (GST).
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned protein of interest can be released from the GST moiety at will.
  • yeast Saccharomyces cerevisiae
  • a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH
  • sequences encoding HRM may be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu (1987) EMBO J 6:307-311).
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi et al. (1984) EMBO J 3:1671-1680; Broglie et al. (1984) Science 224:838-843; and Winter et al. (1991) Results Probl Cell Differ 17:85-105).
  • constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection.
  • Such techniques are described in a number of generally available reviews (see, for example, Hobbs or Murry, hi: McGraw Hill Yearbook of Science and Technolog (1992) McGraw Hill, New York N.Y.; pp. 191-196).
  • An insect system may also be used to express HRM.
  • HRM Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the sequences encoding HRM may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of HRM will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses may then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which HRM may be expressed (Engelhard et al. (1994) Proc Nat Acad Sci 91:3224-3227).
  • a number of viral-based expression systems may be utilized.
  • sequences encoding HRM may be ligated into an adenovirus transcription/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 a viable virus which is capable of expressing HRM in infected host cells (Logan and Shenk (1984) Proc Natl Acad Sci 81:3655-3659).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • HACs Human artificial chromosomes
  • HACs may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
  • HACs of 6 to 10 M are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding HRM. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding HRM, its initiation codon, and upstream sequences are inserted into the expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are for the particular cell system which is used, such as those described in the literature (Scharf et al. (1994) Results Probl Cell Differ 20:125-162).
  • a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the protein include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a “prepro” form of the protein may also be used to facilitate correct insertion, folding and/or function.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the ATCC (Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • cell lines which stably express HRM may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be proliferated using tissue culture techniques 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 (Wigler et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy et al. (1980) Cell 22:817-23) genes which can be employed in tk- or aprt-cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler et al.
  • npt which confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al (1981) J Mol Biol 150:1-14); and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman and Mulligan (1988) Proc Natl Acad Sci 85:8047-51).
  • marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed.
  • sequence encoding HRM is inserted within a marker gene sequence
  • transformed cells containing sequences encoding HRM can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding HRM 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.
  • host cells which contain the nucleic acid sequence encoding HRM and express HRM 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 hybridizations 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.
  • the presence of polynucleotides encoding HRM can be detected by DNA-DNA or DNA-RNA hybridization or PCR amplification.
  • Nucleic acid amplification based assays involve the use of oligonucleotides based on the polynucleotides encoding HRM to detect transformants containing DNA or RNA encoding HRM.
  • a variety of protocols for detecting and measuring the expression of HRM using either polyclonal or monoclonal antibodies specific for the protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on HRM is preferred, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul Minn.) and Maddox et al. (1983; J Exp Med 158:1211-1216).
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding HRM include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide.
  • sequences encoding HRM, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits (APB; Promega, Madison WI).
  • Host cells transformed with polynucleotides encoding HRM may be cultured under conditions for the expression and recovery of the protein from cell culture.
  • the protein produced by a transformed cell may be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode HRM may be designed to contain signal sequences which direct secretion of HRM through a prokaryotic or eukaryotic cell membrane.
  • Other constructions may be used to join sequences encoding HRM to polynucleotide encoding a protein domain which will facilitate purification of soluble proteins.
  • Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex, Seattle WA).
  • metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals
  • protein A domains that allow purification on immobilized immunoglobulin
  • the domain utilized in the FLAGS extension/affinity purification system Immunex, Seattle WA.
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego CA) between the purification domain and HRM may be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing HRM and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site.
  • the histidine residues facilitate purification on IMAC (immobilized metal ion affinity chromatography) as described in Porath et al. (1992, Prot Exp Purif 3:263-281) while the enterokinase cleavage site provides a means for purifying HRM from the fusion protein.
  • IMAC immobilized metal ion affinity chromatography
  • portions of HRM may be produced by direct peptide synthesis using solid-phase techniques (Merrifield (1963) J Am Chem Soc 85:2149-2154). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using 431A Peptide synthesizer (ABI). Various portions of HRM may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.
  • HRM human regulatory proteins of the invention.
  • the expression of HRM is closely associated with cell proliferation. Therefore, in cancers or immune disorders where HRM is an activator, transcription factor, or enhancer, and is promoting cell proliferation; it is desirable to decrease the expression of HRM. In cancers where HRM is an inhibitor or suppressor and is controlling or decreasing cell proliferation, it is desirable to provide the protein or to increase the expression of HRM.
  • HRM is an inhibitor
  • HRM or a portion or derivative thereof may be administered to a subject to treat a cancer
  • a cancer such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma.
  • cancers include, but are not limited to, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.
  • an agonist which is specific for HRM may be administered to a subject to treat a cancer including, but not limited to, those cancers listed above.
  • a vector capable of expressing HRM, or a portion or a derivative thereof may be administered to a subject to treat a cancer including, but not limited to, those cancers listed above.
  • antagonists which decrease the expression or activity of HRM may be administered to a subject to treat a cancer such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma.
  • a cancer such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma.
  • Such cancers include, but are not limited to, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.
  • antibodies which specifically bind HRM may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express HRM.
  • a vector expressing the complement of the polynucleotide encoding HRM may be administered to a subject to treat a cancer including, but not limited to, those cancers listed above.
  • HRM is promoting leukocyte activity or proliferation
  • antagonists which decrease the activity of HRM may be administered to a subject to treat an immune response.
  • Such responses may be associated with AIDS, Addison's disease, adult respiratory distress syndrome, allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitus, Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, atrophic gastritis, glomerulonephritis, gout, Graves' disease, hypereosinophilia, irritable bowel syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis, scleroderma,
  • a vector expressing the complement of the polynucleotide encoding HRM may be administered to a subject to treat an immune response including, but not limited to, those listed above
  • HRM or a portion or derivative thereof may be added to cells to stimulate cell proliferation.
  • HRM may be added to a cell in culture or cells in vivo using delivery mechanisms such as liposomes, viral based vectors, or electroinjection for the purpose of promoting cell proliferation and tissue or organ regeneration.
  • HRM may be added to a cell, cell line, tissue or organ culture in vitro or ex vivo to stimulate cell proliferation for use in heterologous or autologous transplantation.
  • the cell will have been preselected for its ability to fight an infection or a cancer or to correct a genetic defect in ⁇ disease such as sickle cell anemia, , thalassemia, cystic fibrosis, or Huntington's chorea.
  • ⁇ disease such as sickle cell anemia, , thalassemia, cystic fibrosis, or Huntington's chorea.
  • an agonist which is specific for HRM may be administered to a cell to stimulate cell proliferation, as described above.
  • a vector capable of expressing HRM, or a portion or a derivative thereof may be administered to a cell to stimulate cell proliferation, as described above.
  • any of the therapeutic proteins, antagonists, antibodies, agonists, complementary sequences or vectors of the invention may be administered in combination with other therapeutic agents. Selection of the 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 of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • Antagonists or inhibitors of HRM may be produced using methods which are generally known in the art.
  • purified HRM may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind HRM.
  • Antibodies to HRM may be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies, (i.e., those which inhibit dimer formation) are especially preferred for therapeutic use.
  • various hosts including goats, rabbits, rats, mice, humans, and others, may be immunized by injection with HRM or any portion or oligopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • 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, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are especially preferable.
  • the oligopeptides, peptides, or portions used to induce antibodies to HRM have an amino acid sequence consisting of at least five amino acids and more preferably at least 10 amino acids. It is also preferable that they are identical to a portion of the amino acid sequence of the natural protein, and they may contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of HRM amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule.
  • Monoclonal antibodies to HRM 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 hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al. (1975) Nature 256:495-497 Kozbor et al. (1985) J Immunol Methods 81:31-42 Cote et al. (1983) Pr Natl Acad Sci 80:2026-2030, Cole et al. (1984) Mol Cell Biol 62:109-120).
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al. (1989) Proc Natl Acad Sci 86:3833-3837, Winter et al. (1991) Nature 349:293-299).
  • Antibody fragments which contain specific binding sites for HRM may also be generated.
  • fragments include, but are not limited to, the F(ab′)2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab′)2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al. (1989) Science 254:1275-1281).
  • Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between HRM and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering HRM epitopes is preferred, but a competitive binding assay may also be employed (Maddox, supra).
  • the polynucleotides encoding HRM may be used for therapeutic purposes.
  • the complement of the polynucleotide encoding HRM may be used in situations in which it would be desirable to block the transcription of the mRNA.
  • cells may be transformed with sequences complementary to polynucleotides encoding HRM.
  • complementary molecules or fragments may be used to modulate HRM activity, or to achieve regulation of gene function.
  • sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding HRM.
  • Expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of polynucleotides to the targeted organ, tissue or cell population. Methods which are well known to those skilled in the art can be used to construct vectors which will express nucleic acid sequence which is complementary to the polynucleotides of the gene encoding HRM. These techniques are described both in Sambrook (ura and in Ausubel (supra).
  • Genes encoding HRM can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide or fragment thereof which encodes HRM. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector and even longer if replication elements are part of the vector system.
  • modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5′ or regulatory regions of the gene encoding HRM (signal sequence, promoters, enhancers, and introns). Oligonucleotides derived from the transcription initiation site, e.g., between positions ⁇ 10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using “triple helix” base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee et al.
  • the complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples which may be used include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding HRM.
  • RNA target Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • RNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding HRM. Such DNA sequences may be incorporated into a wide variety of vectors with 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.
  • 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 polycationic amino polymers (Goldman et al. (1997) Nature Biotechnol 15:462-66, incorporated herein by reference) may be achieved using methods which are well known in the art.
  • any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • An additional embodiment of the invention relates to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
  • Such pharmaceutical compositions may consist of HRM, antibodies to HRM, mimetics, agonists, antagonists, or inhibitors of HRM.
  • the compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • these pharmaceutical compositions may contain pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Mack Publishing, Easton PA).
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages for oral administration.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding auxiliaries, if desired, to obtain tablets or dragee cores.
  • Excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol, starch from corn, wheat, rice, potato, or other plants, cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose, gums including arabic and tragacanth, and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores may be used in conjunction with coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution. Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds may be prepared as oily injection suspensions.
  • Lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers may also be used for delivery.
  • the suspension may also contain stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • penetrants to the particular barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is com buffer prior to use.
  • compositions After pharmaceutical compositions have been prepared, they can be placed in a container and labeled for treatment of an indicated condition.
  • labeling would include amount, frequency, and method of administration.
  • compositions for use in the invention include compositions wherein the active ingredients are contained in 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.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model may also be used to determine the 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 HRM or portions thereof, antibodies of HRM, agonists, antagonists or inhibitors of HRM, which ameliorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • ED50 the dose therapeutically effective in 50% of the population
  • LD50 the dose lethal to 50% of the population.
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide levels of the active moiety that produce or maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, 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 proteins will be specific to particular cells, conditions, locations, etc.
  • antibodies which specifically bind HRM may be used for the diagnosis of conditions or diseases characterized by expression of HRM, or in assays to monitor patients being treated with HRM, agonists, antagonists or inhibitors.
  • the antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for HRM include methods which utilize the antibody and a label to detect HRM in human body fluids or extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by joining them, either covalently or non-covalently, with a reporter molecule.
  • a wide variety of reporter molecules which are known in the art may be used, several of which are described above.
  • HRM human immunosorbent assay
  • ELISA ELISA
  • RIA RIA
  • FACS fluorescence-activated cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic gammas, cytoplasmic gammas, gammasis, gammasis factor-associated cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic
  • the polynucleotides encoding HRM may be used for diagnostic purposes.
  • the polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of HRM may be correlated with disease.
  • the diagnostic assay may be used to distinguish between absence, presence, and excess expression of HRM, and to monitor regulation of HRM levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding HRM or closely related molecules, may be used to identify nucleic acid sequences which encode HRM.
  • the specificity of the probe whether it is made from a highly specific region, e.g., 10 unique nucleotides in the 5′ regulatory region, or a less specific region, e.g., especially in the 3′ coding region, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low) will determine whether the probe identifies only naturally occurring sequences encoding HRM, alleles, or related sequences.
  • Probes may also be used for the detection of related sequences, and should preferably contain at least 50% of the nucleotides from any of the HRM encoding sequences.
  • the hybridization probes of the invention may be DNA or RNA and derived from the polynucleotide of SEQ ID NOs:50-98 or from genomic sequence including promoter, enhancer elements, and introns of the naturally occurring HRM.
  • Means for producing specific hybridization probes for polynucleotides encoding HRM include the cloning of nucleic acid sequences encoding HRM or HRM derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the RNA polymerases and the labeled nucleotides.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, radionuclides such as 32P or 35S, or enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotides encoding HRM may be used for the diagnosis of conditions, disorders, or diseases which are associated with either increased or decreased expression of HRM.
  • conditions or diseases include adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and cancers of the adrenal gland, bladder, bone, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, bone marrow, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus, and immune disorders such as AIDS, Addison's disease, adult respiratory distress syndrome, allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitus, Crohn's disease, ulcerative colitis, atopic dermatitis, der
  • the polynucleotides encoding HRM may be used in Southern or northern analysis, dot blot, or other membrane-based technologies, in PCR technologies, or in dipstick, pin, or other multiformat assays including microarrays to analyze fluids or tissues from patient biopsies to detect altered HRM expression. Such qualitative or quantitative methods are well known in the art.
  • the polynucleotides encoding HRM may be useful in assays that detect activation or induction of various cancers, particularly those mentioned above.
  • the polynucleotides encoding HRM may be labeled by standard methods, and added to a fluid or tissue sample from a patient under conditions for the formation of hybridization complexes. After an incubation period, the sample is washed, the signal is quantitated and compared with a standard value.
  • the polynucleotides have hybridized with nucleic acids in the sample, and the presence of differentially expressed polynucleotides encoding HRM in the sample indicates the presence of the disease.
  • assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.
  • 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, which encodes HRM, under conditions for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with those from an experiment where a known amount of an isolated polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for disease. Deviation between standard and subject values is used to establish the presence of disease.
  • hybridization assays may be repeated on a regular basis to evaluate whether the level of expression in the patient begins to approximate that which is observed in the normal patient. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of a relatively high amount of transcript 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 prior to the appearance of actual clinical symptoms.
  • a more definitive diagnosis of this type may allow health professionals to employ aggressive treatment earlier thereby preventing further progression of the cancer.
  • oligonucleotides designed from the sequences encoding HRM may involve the use of PCR. Such oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably consist of two polynucleotides, one with sense orientation (5′ ⁇ >3′) and another with antisense (3′ ⁇ 5′), employed under optimized conditions for identification of a specific gene or condition. The same two oligomers, nested sets of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantitation of closely related DNA or RNA sequences.
  • Methods which may also be used to quantitate the expression of HRM include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated (Melby et al. (1993) J Immunol Methods, 159:235-244, Duplaa et al. (1993) Anal Biochem 229-236).
  • the speed of quantitation of multiple samples may be accelerated by running the assay in an multiwell format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • oligonucleotides or longer fragments derived from any of the polynucleotides may be used as targets on a microarray.
  • the microarray can be used to monitor the expression level of large numbers of genes simultaneously (to produce a transcript image), and to identify genetic variants, mutations and polymorphisms. This information may be used to determine gene function, understanding the genetic basis of disease, diagnosing disease, and in developing and in monitoring the activities of therapeutic agents.
  • the microarray is prepared and used according to the methods described in PCT application WO95/11995, Lockhart et al. (1996, Nature Biotechnol 14:1675-1680) and Schena et al. (1996, Proc Natl Acad Sci 93:10614-10619), all of which are incorporated herein in their entirety by reference.
  • the microarray is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support.
  • the oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray, it may be preferable to use oligonucleotides which are only 7-10 nucleotides in length.
  • the microarray may contain oligonucleotides which cover the known 5′, or 3′, sequence, or contain sequential oligonucleotides which cover the full length sequence, or unique oligonucleotides selected from particular areas along the length of the sequence.
  • Polynucleotides used in the microarray may be oligonucleotides that are specific to a gene or genes of interest in which at least a fragment of the sequence is known or that are specific to one or more unidentified cDNAs which are common to a particular cell or tissue type or to a normal, developmental, or disease state. In certain situations it may be to use pairs of oligonucleotides on a microarray.
  • the “pairs” will be identical, except for one nucleotide which is located in the center of the sequence.
  • the second oligonucleotide in the pair (mismatched by one) serves as a control.
  • the number of oligonucleotide pairs may range from 2 to one million.
  • the gene of interest is examined using a computer algorithm which starts at the 5′ or more preferably at the 3′ end of the polynucleotide.
  • the algorithm identifies oligomers of defined length that are unique to the gene, have a GC content within a range for hybridization, and lack predicted secondary structure that may interfere with hybridization.
  • the oligomers are synthesized at designated areas on a substrate using a light-directed chemical process.
  • the substrate may be paper, nylon or other type of membrane, filter, chip, glass slide, or any other solid support.
  • the oligonucleotides may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference.
  • a gridded array analogous to a dot or slot blot apparatus may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
  • an array may be produced by hand or using available devices, materials, and machines (including multichannel pipetters or robotic instruments) and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other multiple from 2 to one million which lends itself to the efficient use of commercially available instrumentation.
  • polynucleotides are extracted from a biological sample.
  • the biological samples may be obtained from any bodily fluid (blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations.
  • the polynucleotides extracted from the sample are used to produce nucleic acid sequences which are complementary to the nucleic acids on the microarray. If the microarray consists of cDNAs, antisense RNAs (aRNA) are probes.
  • aRNA antisense RNAs
  • mRNA is used to produce cDNA which, in turn and in the presence of fluorescent nucleotides, is used to produce fragment or oligonucleotide aRNA probes. These fluorescently labeled probes are incubated with the microarray so that the probe sequences hybridize to the cDNA oligonucleotides of the microarray.
  • complementary nucleic acid sequences are used as probes and can also include polynucleotides, fragments, complementary, or antisense sequences produced using restriction enzymes, PCR technologies, and oligolabeling kits which are well known in the art.
  • Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity.
  • a scanner is used to determine the levels and patterns of fluorescence.
  • the scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray.
  • a detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large scale correlation studies or functional analysis of the sequences, mutations, variants, or polymorphisms among samples (Heller et al. (1997) Proc Natl Acad Sci 94:2150-55).
  • the nucleic acid sequences which encode HRM may also be used to generate hybridization probes which are useful for mapping the naturally occurring genomic sequence.
  • the sequences may be mapped to a particular chromosome, to a specific region of a chromosome or to artificial chromosome constructions, such as human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial chromosome cDNA libraries as reviewed in Price (1993, Blood Rev 7:127-134) and Trask (1991, Trends Genet 7:149-154).
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDNA libraries as reviewed in Price (1993, Blood Rev 7:127-134) and Trask (1991, Trends Genet 7:149-154).
  • Fluorescent in situ hybridization may be correlated with other physical chromosome mapping techniques and genetic map data. Examples of genetic map data can be found in various scientific journals or at Online Mendelian Inheritance in Man (OMIM). Correlation between the location of the gene encoding HRM on a physical chromosomal map and a specific disease, or predisposition to a specific disease, may help delimit the region of DNA associated with that genetic disease.
  • the polynucleotides of the invention may be used to detect differences in gene sequences between normal, carrier, or affected individuals.
  • In situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms, or parts thereof, by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, for example, AT to 11q22-23 (Gatti et al.
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the polynucleotide of the invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals.
  • HRM its catalytic or immunogenic portions or oligopeptides thereof
  • the portion employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes, between HRM and the agent being tested, may be measured.
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having binding affinity to the protein of interest as described in published PCT application WO84/03564.
  • a solid substrate such as plastic pins or some other surface.
  • the test compounds are reacted with HRM, or portions thereof, and washed. Bound HRM is then detected by methods well known in the art.
  • Purified HRM 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 polynucleotides which encode HRM may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of polynucleotides that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
  • the LNODNOT03 cDNA library was constructed using 1 ⁇ g of polyA RNA isolated from lymph node tissue removed from a 67-year-old Caucasian male during a segmental lung resection and bronchoscopy. Microscopic examination showed that the tissue was extensively necrotic with 10% viable tumor. The invasive grade 3/4 squamous cell carcinoma had formed a mass in the right lower lobe of the lung which had invaded into, but not through, the visceral pleura. Focally, the tumor had obliterated the bronchial lumen although the bronchial margin was negative for dysplasia/neoplasm.
  • One of two intrapulmonary, one of four inferior mediastinal (subcarinal), and two of eight superior mediastinal lymph nodes were metastatically involved.
  • Patient history included hemangioma and tobacco use, the patient was taking Doxycycline, a tetracycline, to treat an infection.
  • the frozen tissue was homogenized and lysed in guanidinium isothiocyanate solution using a POLYTRON homogenizer (Brinkmann Instruments, Westbury N.Y.). The lysate was centrifuged over a 5.7 M CsCl cushion using an SW28 rotor in a L8-70M ultracentrifuge (Beckman Coulter, Fullerton Calif.) for 18 hours at 25,000 rpm at ambient temperature. The RNA was extracted with acid phenol, pH 4.7, precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended in RNAse-free water, and treated with DNAse at 37C. Extraction and precipitation were repeated as before. The MRNA was isolated using the OLIGOTEX kit (Qiagen, Chatsworth Calif.) and used to construct the cDNA library.
  • the mRNA was handled according to the recommended protocols in the SUPERSCRIPT plasmid system (Life Technologies).
  • the cDNAs were fractionated on a SEPHAROSE CL4B column (APB), and those cDNAs exceeding 400 bp were ligated into pINCY plasmid (Incyte Genomics).
  • the plasmid was subsequently transformed into DH5 ⁇ competent cells (Life Technologies).
  • Plasmid DNA was released from the cells and purified using the REAL Prep 96 plasmid kit (Qiagen). This kit enabled the simultaneous purification of 96 samples in a 96-well block using multi-channel reagent dispensers.
  • the recommended protocol was employed except for the following changes: 1) the bacteria were cultured in 1 ml of sterile TERRIFIC BROTH (BD Biosciences, Sparks MD) with carbenicillin at 25 mg/L and glycerol at 0.4%, 2) after incubation for 19 hours, the cells were lysed with 0.3 ml of lysis buffer and precipitated using isopropanol, and 3) the plasmid pellet was resuspended in 0.1 ml of distilled water. After the last step in the protocol, samples were transferred to a 96-well block for storage at 4 C.
  • cDNAs were prepared using a MICROLAB system (Hamilton) in combination with DNA ENGINE thermal cyclers (MJ Research), sequenced by the method of Sanger and Coulson (1975, J Mol Biol 94:441f) using 377 PRISM DNA sequencing systems (ABI), and reading frame was determined.
  • BLAST produced alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST was especially useful in determining exact matches or in identifying homologs which may be of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant) origin. Other algorithms such as the one described in Smith et al. (1992, Protein Engineering 5:35-51) could have been used when dealing with primary sequence patterns and secondary structure gap penalties. The sequences disclosed in this application have lengths of at least 49 nucleotides, and no more than 12% uncalled bases (where N is recorded rather than A, C, G, or T).
  • BLAST approach searched for matches between a query sequence and a database sequence. BLAST evaluated the statistical significance of any matches found and reported only those matches that satisfy the user-selected threshold of significance. In this application, threshold was set at 10 ⁇ 25 for nucleotides and 10 ⁇ 14 for peptides.
  • Incyte polynucleotides were searched against the GenBank databases for primate (pri), rodent (rod), and other mammalian sequences (mam), and deduced amino acid sequences from the same clones were then searched against GenBank functional protein databases, mammalian (mamp), vertebrate (vrtp), and eukaryote (eukp) for homology.
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled polynucleotide to a membrane on which RNAs from a particular cell type or tissue have been bound (Sambrook, supra).
  • Analogous computer techniques use BLAST to search for identical or related molecules in nucleotide databases such as GenBank or the LIFESEQ database (Incyte Genomics). This analysis is much faster than multiple, membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or homologous.
  • the product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1-2% error, and at 70, the match will be exact. Homologous molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.
  • nucleic acid sequence of an Incyte Clone disclosed in the Sequence Listing was used to design oligonucleotide primers for extending a partial sequence to full length.
  • One primer was synthesized to initiate extension in the antisense direction, and the other was synthesized to extend sequence in the sense direction.
  • Primers were used to facilitate the extension of the known sequence “outward” generating amplicons containing new, unknown nucleotide sequence for the region of interest.
  • the initial primers were designed from the cDNA using OLIGO software (Molecular Insights), or another program, to be about 22 to about 30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures of about 68 to about 72 C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
  • Step 1 For PCR amplification, 18 , ⁇ l of concentrated PCR reaction mix (3.3x) containing 4 units of rTth DNA polymerase, a vector primer, and one or both of the gene specific primers used for the extension reaction were added to each well. Amplification was performed using the following conditions: Step 1, 94 C for 60 sec; Step 2, 94 C for 20 sec; Step 3, 55 C for 30 sec; Step 4, 72 C for 90 sec; Step 5, repeat steps 2-4 for an additional 29 cycles, Step 6, 72 C for 180 sec, and Step 7, hold at 4 C.
  • Hybridization probes derived from SEQ ID NOs:50-98 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base-pairs, is specifically described, the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO software (Molecular Insights), labeled by combining 50 pmol of each oligomer and 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (APB) and T4 polynucleotide kinase (NEN Life Science Products, Acton Mass.).
  • state-of-the-art software such as OLIGO software (Molecular Insights)
  • APB adenosine triphosphate
  • T4 polynucleotide kinase NN Life Science Products, Acton Mass.
  • the labeled oligonucleotides are purified using SEPHADEX G-25 superfine resin column (APB). A aliquot containing 10 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II, NEN Life Science Products).
  • oligonucleotides for a microarray SEQ ID NOs:50-98 are examined using a computer algorithm which starts at the 3′ end of the polynucleotide.
  • the algorithm identified oligomers of defined length that are unique to the gene, have a GC content within a range for hybridization, and lack predicted secondary structure that would interfere with hybridization.
  • the algorithm identifies approximately 20 sequence-specific oligonucleotides of 20 nucleotides in length (20-mers).
  • a matched set of oligonucleotides are created in which one nucleotide in the center of each sequence is altered. This process is repeated for each gene in the microarray, and double sets of twenty 20 mers are synthesized and arranged on the surface of the silicon chip using a light-directed chemical process (described in PCT/WO95/11995).
  • a chemical coupling procedure and an ink jet device are used to synthesize oligomers on the surface of a substrate (PCT/WO95/251116).
  • a gridded array is used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical, or chemical bonding procedures.
  • a typical array may be produced by hand or using available materials and machines and contain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots.
  • the microarray is washed to remove nonhybridized probes, and a scanner is used to determine the levels and patterns of fluorescence. The scanned image is examined to determine degree of complementarity and the relative abundance/expression level of each sequence in the microarray.
  • Sequence complementary to the sequence encoding HRM, or any part thereof, is used to detect, decrease or inhibit expression of naturally occurring HRM.
  • oligonucleotides comprising from about 15 to about 30 base-pairs is described, the same procedure is used with smaller or larger sequence fragments.
  • Oligonucleotides are designed using OLIGO software (Molecular Insights) and the coding sequence of SEQ ID NOs:50-98.
  • OLIGO software Molecular Insights
  • a complementary oligonucleotide is designed from the most unique 5′ sequence and used to prevent promoter binding to the coding sequence.
  • a complementary oligonucleotide is designed to prevent ribosomal binding to the transcript encoding HRM.
  • HRM expression of HRM is accomplished by subcloning the cDNAs into vectors and transforming the vectors into host cells.
  • the cloning vector is also used to express HRM in E. coli. Upstream of the cloning site, this vector contains a promoter for ⁇ -galactosidase, followed by sequence containing the amino-terminal Met, and the subsequent seven residues of ⁇ -galactosidase. Immediately following these eight residues is a bacteriophage promoter useful for transcription and a linker containing a number of unique restriction sites.
  • Induction of an isolated, transformed bacterial strain with IPTG using standard methods produces a fusion protein which consists of the first eight residues of ⁇ -galactosidase, about 5 to 15 residues of linker, and the full length protein.
  • the signal residues direct the secretion of HRM into the bacterial growth media which can be used directly in the following assay for activity.
  • HRM can be expressed in a mammalian cell line such as DLD-1 or HCT116 (ATCC) by transforming the cells with a eukaryotic expression vector encoding HRM.
  • Eukaryotic expression vectors are commercially available and the techniques to introduce them into cells are well known to those skilled in the art.
  • the effect of HRM on cell morphology may be visualized by microscopy, the effect on cell growth may be determined by measuring cell doubling-time, and the effect on tumorigenicity may be assessed by the ability of transformed cells to grow in a soft agar growth assay (Groden (1995) Cancer Res 55:1531-1539).
  • HRM that is purified using PAGE electrophoresis (Sambrook, supra), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
  • an amino acid sequence deduced from SEQ ID NOs:50-98 is analyzed using LASERGENE software (DNASTAR, Madison Wis.) to determine regions of high immunogenicity, and an oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Selection of epitope, such as those near the C-terminus or in hydrophilic regions, is described by Ausubel (supra).
  • the oligopeptides are 15 residues in length, synthesized using a 43 1A Peptide synthesizer (ABI) using Fmoc-chemistry, and coupled to keyhole limpet hemocyanin (Sigma-Aldrich, St. Louis Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (Ausubel supra). Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. The resulting antisera are tested for antipeptide activity, for example, by binding the protein to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio iodinated, goat anti-rabbit IgG.
  • ABSI 43 1A Peptide synthesizer
  • Naturally occurring or recombinant HRM is substantially purified by immunoaffinity chromatography using antibodies specific for HRM.
  • An immunoaffinity column is constructed by covalently coupling HRM antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE resin (APB). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
  • HRM or biologically active portions thereof, are labeled with 125 I Bolton-Hunter reagent (Bolton et al. (1973) Biochem J 133:529-39).
  • Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled HRM, washed and any wells with labeled HRM complex are assayed. Data obtained using different concentrations of HRM are used to calculate values for the number, affinity, and association of HRM with the candidate molecules.

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Abstract

The invention provides human regulatory molecules and the polynucleotides which identify and encode them. The invention also provides expression vectors, host cells, agonists, antibodies and antagonists. The invention further provides methods for diagnosing and treating disorders associated with expression of human regulatory molecules.

Description

  • This application is a divisional of U.S. Pat. No. 09/518,865 filed 3 Mar. 2000, which was a divisional of U.S. Pat. No 6,132,973, issued 17 Oct. 2000, which was a divisional of U.S. Pat. No. 5,932,442, issued 3 Aug. 1999.[0001]
    • FIELD OF THE INVENTION
  • This invention relates to nucleic acid and amino acid sequences of human regulatory molecules which are implicated in disease and to the use of these sequences in the diagnosis and treatment of diseases associated with cell proliferation. [0002]
    • BACKGROUND OF THE INVENTION
  • Cells grow and differentiate, carry out their structural or metabolic roles, participate in organismal development, and respond to their environment by altering their gene expression. Cellular functions are controlled by the timing and amount of expression attributable to thousands of individual genes. The regulation of expression is metabolically vital in that it conserves energy and prevents the synthesis and accumulation of intermediates such as RNA and incomplete or inactive proteins when the gene product is not needed. [0003]
  • Regulatory protein molecules function to control gene expression. These molecules turn individual or groups of genes on and off in response to various inductive mechanisms of the cell or organism; act as transcription factors by determining whether or not transcription is initiated, enhanced, or repressed; and splice transcripts as dictated in a particular cell or tissue. Although regulatory molecules interact with short stretches of DNA scattered throughout the entire genome, most gene expression is regulated near the site at which transcription starts or within the open reading frame of the gene being expressed. The regulated stretches of the DNA can be simple and interact with only a single protein, or they can require several proteins acting as part of a complex in order to regulate gene expression. [0004]
  • The double helix structure and repeated sequences of DNA create external features which can be recognized by the regulatory molecules. These external features are hydrogen bond donor and acceptor groups, hydrophobic patches, major and minor grooves, and regular, repeated stretches of sequence which cause distinct bends in the helix. Such features provide recognition sites for the binding of regulatory proteins. Typically, these recognition sites are less than 20 nucleotides in length although multiple sites may be adjacent to each other, and each may exert control over a single gene. Hundreds of these DNA sequences have been identified, and each is recognized by a different protein or complex of proteins which carry out gene regulation. [0005]
  • The regulatory protein molecules or complexes recognize and bind to specific nucleotide sequences of upstream (5′) nontranslated regions, which precede the first translated exon of the open reading frame (ORF); of intron junctions, which occur between the many exons of the OR; and of downstream (3′) untranslated regions, which follow the ORF. The regulatory molecule surface features are extensively complementary to the surface features of the double helix. Even though each individual contact between the protein(s) and helix may be relatively weak (hydrogen bonds, ionic bonds, and/or hydrophobic interactions) and the 20 or more contacts occurring between the protein and DNA result in a highly specific and very strong interaction. [0006]
    • Families of regulatory molecules
  • Many of the regulatory molecules incorporate one of a set of DNA-binding structural motifs, each of which contains either α helices or β sheets and binds to the major groove of DNA. Seven of the structural motifs common to regulatory molecules are helix-turn-helix, homeodomains, zinc finger, steroid receptor, β sheets, leucine zipper, and helix-loop-helix. [0007]
  • The helix-turn-helix motif is constructed from two α helices connected by a short chain of amino acids, which constitutes the “turn”. The two helices interact with each other to form a fixed angle. The more carboxy-terminal helix is called the recognition helix because it fits into the major groove of the DNA. The amino acid side chains of the helix recognize the specific DNA sequence to which the protein binds. The H remaining structure varies a great deal among the regulatory proteins incorporating this motif. The helix-turn-helix configuration is not stable without the rest of the protein and will not bind to DNA without other peptide regions providing stability. Other peptide regions also interact with the DNA, increasing the number of unique sequences a helix-turn-helix can recognize. [0008]
  • Many sequence-specific DNA binding proteins actually bind as symmetric dimers to DNA sequences that are composed of two very similar half-sites, also arranged symmetrically. This configuration allows each protein monomer to interact in the same way with the DNA recognition site and doubles the number of contacts with the DNA. This doubling of contacts greatly increases the binding affinity while only doubling the free energy of the interaction. Helix-turn-helix motifs always bind to DNA that is in the B-DNA form. [0009]
  • The homeodomain motif is found in a special group of helix-turn-helix proteins that are encoded by homeotic selector genes, so called because the proteins encoded by these genes control developmental switches. For example, mutations in these genes cause one body part to be converted into another in the fruit fly, Drosophila. These genes have been found in every eukaryotic organism studied. The helix-turn-helix region of different homeodomains is always surrounded by the same structure, but not necessarily the same sequence, and the motif is always presented to DNA the same way. This helix-turn-helix configuration is stable by itself and, when isolated, can still bind to DNA. It may be significant that the helices in homeodomains are generally longer than the helices in most HLH regulatory proteins. Portions of the motif which interact most directly with DNA differ among these two families. Detailed examples of DNA-protein binding are described in Pabo and Sauer (1992; Ann Rev Biochem 61:1053-95). [0010]
  • A third motif incorporates zinc molecules into the crucial portion of the protein. These proteins are most often referred to as having zinc fingers, although their structure can be one of several types. Proteins in this family often contain tandem repeats of the 30-residue zinc finger motif, including the sequence patterns Cys-X[0011] 2 or 4-Cys-X12-His-X3--His. Each of these regulatory proteins has an a helix and an antiparallel β sheet. Two histidines in the α helix and 2 cysteines near the turn in the β sheet interact with the zinc ion which holds the α helix and the β sheet together. Contact with the DNA is made by the arginine preceding the α helix, and by the second, third, and sixth residues of the α helix. When this arrangement is repeated as a cluster of several fingers, α of each finger can contact and interact with the major groove of the DNA. By changing the number of zinc fingers, the specificity and strength of the binding interaction can be altered.
  • The steroid receptors are a family of intracellular proteins that include receptors for steroids, retinoids, vitamin D, thyroid hormones, and other important compounds. The DNA binding domain of these proteins contains about 70 residues, eight of which are conserved cysteines. The steroid receptor motif forms a structure in which two a helices are packed perpendicularly to each other, forming more of a globular shape than a finger. Each helix has a zinc ion which holds a peptide loop against the N-terminal end of the helix. The first helix fits into the major groove of DNA, and side chains make contacts with edges of the DNA base pairs. The steroid receptor proteins, like the helix-turn-helix proteins, form dimers that bind the DNA. The second helix of each monomer contacts the phosphate groups of the DNA backbone and also provides the dimerization interface. In some cases, multiple choices can exist for heterodimerization which produces another mechanism for fine-tuning the regulation of numerous genes. [0012]
  • Another family of regulatory protein molecules uses a motif consisting of a two-stranded antiparallel βsheet to recognize the major groove of DNA. The exact DNA sequence recognized by the motif depends on the amino acid sequence in the β sheet from which the amino acid side chains extend and contact the DNA. In two prokaryotic examples of the β sheet, the regulatory proteins form tetramers when binding DNA. [0013]
  • The leucine zipper motif commonly forms dimers and has a 30-40 residue motif in which two α helices (one from each monomer) are joined to form a short coiled-coil. The helices are held together by interactions among hydrophobic amino acid side chains (often on heptad-repeated leucines) that extend from one side of each helix. Beyond this, the helices separate, and each basic region contacts the major groove of DNA. Proteins with the leucine zipper motif can also form either homodimers or heterodimers, thus extending the specific combinations available to activate or repress expression. [0014]
  • Yet another motif is the helix-loop-helix, which consists of a short α helix connected by a loop to a longer α helix. The loop is flexible and allows the two helices to fold back against each other. The α helices bind both to DNA and to the HLH structure of another protein. The second protein can be the same (producing homodimers) or different (producing heterodimers). Some HLH monomers lack sufficient α helix to bind DNA, but they can still form heterodimers which can serve to inactivate specific regulatory proteins. [0015]
  • Hundreds of regulatory proteins have been identified to date, and more are being characterized in a wide variety of organisms. Most regulatory proteins have at least one of the common structural motifs for making contact with DNA, but several regulatory proteins, such as the p53 tumor suppressor gene, do not share their structure with other known regulatory proteins. Variations on the known motifs and new motifs have been and are currently being characterized (Faisst and Meyer (1992) Nucl Acids Res 20:3-26). [0016]
  • Although binding of DNA to a regulatory protein is very specific, there is no way to predict the exact DNA sequence to which a particular regulatory protein will bind or the primary structure of a regulatory protein for a specific DNA sequence. Thus, interactions of DNA and regulatory proteins are not limited to the motifs described above. Other domains of the proteins often form crucial contacts with the DNA, and accessory proteins can provide interactions which may convert a particular protein complex to an activator or a represser or may prevent binding (Alberts et al. (1994)[0017] Molecular Biology of the Cell, Garland Publishing, New York NY, pp.401-74).
    • Diseases and disorders related to gene regulation
  • Many neoplastic growths in humans can be traced to problems of gene regulation. Malignant growth of cells may be the result of excess transcriptional activator or loss of an inhibitor or suppressor (Cleary (1992) Cancer Surv 15:89-104). Alternatively, gene fusion may produce chimeric loci with switched domains, such that the level of activation is no longer correct for the gene specificity of that factor. [0018]
  • The cellular response to infection or trauma is beneficial when genes are appropriately expressed. However, when hyper-responsivity or another imbalance occurs for any reason, disregulation of gene expression may cause considerable tissue or organ damage. This damage is well documented in immunological responses to allergens, heart attack, stroke, and infections ([0019] Harrison's Principles of Internal Medicine, 13/e©, (1994) McGraw Hill and Teton Data Systems, Jackson Wyo.). In addition, the accumulation of somatic mutations and the increasing inability to regulate cellular responses is seen in the prevalence of osteoarthritis and onset of other aging disorders.
  • The discovery of new human regulatory protein molecules which are expressed during disease development and the polynucleotides which encode them satisfies a need in the art by providing compositions which are useful in the diagnosis and treatment of diseases associated with cell proliferation, particularly immune responses and cancers. [0020]
    • SUMMARY OF THE INVENTION
  • The invention features purified proteins, human regulatory molecules, collectively referred to as HRM and individually referred to as HRM-1 through HRM-49. In one embodiment, the purified protein comprises an amino acid sequence selected from SEQ ID NO:1 through SEQ ID NO:49 and portions thereof.. [0021]
  • The invention provides isolated polynucleotides encoding HRM and complements of the encoding polynucleotides. In one embodiment, the polynucleotide comprises a nucleic acid sequence selected from SEQ ID NOs:50-98 and complements thereof. [0022]
  • The invention also provides a polynucleotide, or a complement or a fragment thereof, which is used as a probe to hybridize to any one of the polynucleotides of SEQ ID NOs:50-98. The invention further provides a composition comprising the isolated and purified polynucleotides of SEQ ID NOs:50-98. In addition, the invention provides a composition comprising a polynucleotide selected from SEQ ID NOs:50-98 and complements and fragments thereof and a reporter molecule or stabilizing moiety. The invention still further provides a method for detecting expression of a polynucleotide which encodes a human regulatory molecule in a sample, the method comprising hybridizing the complement of a polynucleotide encoding HRM to nucleic acids of the sample under conditions to form a hybridization complex; and detecting hybridization complex formation, wherein complex formation indicates the expression of the polynucleotide encoding the human regulatory molecule in the sample. In one aspect, the complement of the polynucleotide encoding HRM is immobilized on a substrate. In another aspect, the substrate is a microarray. [0023]
  • The invention provides a vector containing at least a fragment of any one of the polynucleotides selected from SEQ ID NOs:50-98. In one embodiment, the vector is contained within a host cell. The invention also provides a method for producing a protein or a portion thereof, the method comprising culturing a host cell containing a vector containing at least a fragment of a polynucleotide encoding an HRM under conditions for the expression of the protein; and recovering the protein from the host cell culture. [0024]
  • The invention further provides a composition comprising a purified HRM and a labeling moiety or a pharmaceutical carrier. The invention still further provides a method for using an HRM to screen a plurality of molecules in order to obtain a ligand which specifically binds the HRM, the method comprising combining the protein with the molecules under conditions which allow specific binding, recovering the bound protein, separating the protein, thereby obtaining the ligand. In one aspect, the molecules are selected from libraries of agonists, antibodies, antagonists, drugs, inhibitors, peptides, proteins, and pharmaceutical agents. [0025]
  • The invention still further provides a method for using a protein to produce and purify an antibody, the method comprising immunizing a animal with an HRM under conditions to elicit an antibody response; isolating animal antibodies; attaching the protein to a substrate; contacting the substrate with sera containing antibodies under conditions to allow specific binding to the HRM; dissociating the antibodies from the HRM, thereby obtaining purified antibodies. [0026]
  • The invention provides a purified antibody which specifically binds an HRM. The invention also provides a method for using an antibody to detect protein expression in a sample, the method comprising combining the antibody specifically binding HRM with a sample under conditions to form antibody:protein complexes and detecting complex formation, wherein detection indicates expression of the protein in the sample. In one aspect, expression of the HRM is diagnostic of cancer. In another aspect, expression is diagnostic of immune response. [0027]
  • The invention also provides a method for diagnosing a disease associated with gene expression in a sample containing nucleic acids, the method comprising hybridizing a polynucleotide to nucleic acids of the sample under conditions to form a hybridization complex, comparing hybridization complex formation to standards, thereby diagnosing the disease. In one aspect, the disease is selected from a disorder characterized by cell proliferation such as a cancer, an developmental disorder, or an immune response. [0028]
  • The invention provides a method for treating a cancer comprising administering to a subject in need of such treatment a composition containing purified HRM. The invention also provides a method for treating a cancer comprising administering to a subject in need of such treatment an antagonist which specifically binds HRM. The invention further provides a method for treating an immune response associated with the increased expression or activity of HRM comprising administering to a subject in need of such treatment an antagonist which specifically binds HRM. The invention still further provides a method for stimulating cell proliferation comprising administering purified HRM to a cell. [0029]
    • DESCRIPTION OF THE INVENTION
  • Before the present proteins, polynucleotide, and methods are described, it is to be understood that this invention is not limited to any particular methodology, protocol, cell line, vector, or reagent, as these may vary. It must also be noted that in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. For example, reference to “a host cell” includes a plurality of such host cells; reference to an “antibody” includes one or more antibodies and equivalents thereof known to those skilled in the art. [0030]
  • Unless defined herein, all technical and scientific terms have the same meanings commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology is used for the purpose of describing particular embodiments and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Although any methods and materials similar or equivalent to those described can be used in the practice or testing of the invention, the preferred methods, devices, and materials are now described. All publications are incorporated herein by reference for the purpose of describing and disclosing the cell lines, vectors, arrays and methodologies which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. [0031]
  • DEFINITIONS [0032]
  • “Agonist” refers to a molecule which specifically binds to and modulates the activity of HRM. [0033]
  • An “allele” is an alternative form of the polynucleotide or gene encoding HRM. Alleles result from at least one mutation in the nucleic acid sequence and may result in the expression of altered mRNAs or proteins whose structure or function may or may not be altered. Any given gene may have none, one, or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to additions, deletions, 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. Similarly a polynucleotide may be altered to produce deliberate amino acid substitutions. Theses substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological or immunological activity of HRM is retained. For example, negatively charged residues include aspartic acid and glutamic acid; positively charged residues include lysine and arginine; and residues with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, and valine, glycine and alanine, asparagine and glutamine, serine and threonine, and phenylalanine and tyrosine. [0034]
  • “Antagonist” refers to a molecule which, when bound to HRM, decreases the amount or the duration of the biological or immunological activity of HRM. Antagonists may include proteins, nucleic acids, carbohydrates, fats or any other molecules which decrease the effect of HRM. [0035]
  • “Antibody” refers to intact molecules, or fragments thereof such as Fa, F(ab′)[0036] 2, and Fv, which are capable of binding the antigenic determinant of an HRM.
  • “Biologically active” refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, “immunologically active” refers to the capability of the natural, recombinant, or synthetic protein or peptide to induce a specific immune response in animals or cells and to bind with specific antibodies. [0037]
  • “Complementary” refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence “A-G-T” binds to the complementary sequence “T-C-A”. The degree of complementarity between nucleic acids has significant effects on the efficiency and strength of hybridization. This is important in amplification reactions and in the design and use of peptide nucleic acid molecules. [0038]
  • A “composition” refers to a combination comprising a plurality of polynucleotides or a specific polynucleotide or protein and at least one other molecule. Such other molecules may include reporter molecules, labeling moieties, pharmaceutical carriers, carbohydrates, and the like. [0039]
  • “Consensus” refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, has been extended using XL-PCR kit (Applied Biosystems (ABI), Foster City Calif.) in the 5′ and/or the 3′ direction and resequenced, or has been assembled to full length from overlapping shorter fragments using a computer program for fragment assembly such as that described in U.S. Ser. No. 09/276,534, filed 25 Mar. 1999. [0040]
  • “Derivative” refers to the chemical modification of a polynucleotide or protein. Such modifications may include replacement of hydrogen by an alkyl, acyl, or amino group. A nucleic acid derivative may encode a protein which retains the biological or immunological function of the natural molecule. A derivative protein is one which is modified by glycosylation, pegylation, or any similar process but still retains the biological or immunological function of the native protein. [0041]
  • “Differential expression” refers to an increased, upregulated or present, or decreased, downregulated or absent, gene expression as detected by presence, absence or at least about two-fold changes in the amount of transcribed messenger RNA or translated protein in a sample. [0042]
  • “Disorder” refers to a condition, disease or syndrome in which a polynucleotide or a protein of the invention is differentially expressed. Such a disorder includes cancers or immune responses as they are set forth below. [0043]
  • “HRM” refers to any one or all of the human proteins, HRMs 1-49, as it was obtained from any species including bovine, ovine, porcine, murine, equine, and preferably human, or from any source whether natural, synthetic, semi-synthetic, or recombinant. [0044]
  • “Hybridization complex” refers to a complex formed between two nucleic acids by the formation of hydrogen bonds between complementary base pairs; these hydrogen bonds form in an antiparallel configuration and may be further stabilized by base stacking interactions. A hybridization complex may be formed in solution or between one nucleic acid present in solution and another nucleic acid immobilized on a substrate. [0045]
  • “Isolated” refers to a polynucleotide that is removed from its natural environment or separated from other components with which it is naturally associated. [0046]
  • “Ligand” refers to any agent, molecule, or compound which will bind specifically to a polynucleotide or to a protein. Such ligands stabilize or modulate the activity of polynucleotides or proteins and may be composed of inorganic and/or organic substances including minerals, cofactors, nucleic acids, proteins, carbohydrates, fats, and lipids. [0047]
  • “Microarray” refers to an arrangement of distinct polynucleotides on a substrate “Oligonucleotide” refers to a nucleic acid sequence about 6 nucleotides to about 60 nucleotides in length which may be used in amplification or hybridization assays. Equivalent terms include “amplimers”,“primers”, “oligomers”, and “probes”, as these are commonly defined in the art. “Peptide nucleic acid” refers to an anti-gene agent which comprises an oligonucleotide of at least five nucleotides in length linked to a peptide backbone of amino acid residues which ends in a terminal lysine which confers solubility to the molecule. [0048]
  • “Polynucleotide” refers to nucleic acid molecule having a nucleic acid sequence and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded and represent the sense or antisense strand. “Fragment” refers to a nucleic acid sequence which is more than about 60 nucleotides in length. [0049]
  • “Portion” refers to a fragment of an HRM which ranges in size from five amino acid residues to the entire amino acid sequence minus one amino acid. [0050]
  • “Protein” refers to an oligopeptide, peptide, or polypeptide having an amino acid sequence whether naturally occurring or synthetic molecules. Portions of HRM are preferably about 5 to about 15 amino acids in length and retain the biological or the immunological activity of the HRM. [0051]
  • “Purified” refers to a peptide or protein that is removed from its natural environment, isolated or separated from other components with which it is naturally associated. [0052]
  • “Reporter molecules” or “labeling moieties” include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like. [0053]
  • “Sample” is used in its broadest sense and may comprise a bodily fluid, extract from a cell, chromosome, organelle, or membrane isolated from a cell, a cell, genomic DNA, RNA, or cDNA (in solution or bound to a solid support), a tissue, a tissue print, and the like. [0054]
  • “Specific binding” refers to that interaction between a polynucleotide or protein of the invention and any ligand which specifically binds to it and which is selected from a DNA or an RNA molecule, a peptide nucleic acid, a peptide, a protein, an agonist, an antibody, an antagonist, an inhibitor, a mimetic, a pharmaceutical agent, a drug, a transcription factor, or an artificial chromosome construction. The interaction is dependent upon the presence of a particular sequence or three dimensional structure recognized by the binding molecule. [0055]
  • “Substrate” refers to any rigid or semi-rigid support to which polynucleotides or proteins are bound and includes membranes, filters, chips, slides, wafers, fibers, magnetic or nomnmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores. [0056]
  • “Variant” refers to molecules that are recognized variations of a polynucleotide or a protein encoded by the polynucleotide. Splice variants may be determined by BLAST score, wherein the score is at least 100, and most preferably at least 400. Allelic variants have a high percent identity to the polynucleotides and may differ by about three bases per hundred bases. “Single nucleotide polymorphism” (SNP) refers to a change in a single base as a result of a substitution, insertion or deletion. The change may be conservative (purine for purine) or non-conservative (purine to pyrimidine) and may or may not result in a change in an encoded amino acid or its secondary, tertiary, or quaternary structure. [0057]
  • THE INVENTION [0058]
  • The invention is based on the discovery of human regulatory molecules (HRM) and the polynucleotides encoding HRM, and on the use of these compositions for the diagnosis and treatment of diseases associated with cell proliferation. Table 1 shows the protein and polynucleotide identification numbers, protein abbreviation, Incyte Clone number, cDNA library, and the closest NCBI homolog and NCBI sequence identifier for each of the human regulatory molecules. [0059]
    TABLE 1
    Protein Nucleotide Abbreviation Clone ID Library NCBI Homolog
    SEQ ID NO:1 SEQ ID NO:50 HRM-1    133 U937NOT01 g285947 KIAA0105
    SEQ ID NO:2 SEQ ID NO:51 HRM-2    1762 U937NOT01 g1518121 Ascaris suum
    SEQ ID NO:3 SEQ ID NO:52 HRM-3    1847 U937NOT01 g1302211 Saccharomyces cerevisiae
    SEQ ID NO:4 SEQ ID NO:53 HRM-4    9337 HMC1NOT01 g1613852 Human zinc finger protein (zf2)
    SEQ ID NO:5 SEQ ID NO:54 HRM-5    9476 HMC1NOT01 g755784 S. cerevisiae
    SEQ ID NO:6 SEQ ID NO:55 HRM-6   10370 THP1PLB01 g895845 Human putative p64 CLCP protein
    SEQ ID NO:7 SEQ ID NO:56 HRM-7   30137 THP1NOB01 g1710241 Human clone 23733 mRNA
    SEQ ID NO:8 SEQ ID NO:57 HRM-8   77180 SYNORAB01 g5372 S. cerevisiae
    SEQ ID NO:9 SEQ ID NO:58 HRM-9   98974 PITUNOR01 g1627704 Caenorhabditis elegans
    SEQ ID NO:10 SEQ ID NO:59 HRM-10  118160 MUSCNOT01 g220594 Mus musculus
    SEQ ID NO:11 SEQ ID NO:60 HRM-11  140516 TLYMNOR01 g1086723 C. elegans
    SEQ ID NO:12 SEQ ID NO:61 HRM-12  207452 SPLNNOT02 g1314086 S. cerevisiae
    SEQ ID NO:13 SEQ ID NO:62 HRM-13  208836 SPLNNOT02 g662126 S. cerevisiae
    SEQ ID NO:14 SEQ ID NO:63 HRM-14  569710 MMLR3DT01 g1698719 Human zinc finger protein
    SEQ ID NO:15 SEQ ID NO:64 HRM-15  606742 BRSTTUT01 g1710201 Human clone 23679 mRNA
    SEQ ID NO:16 SEQ ID NO:65 HRM-16  611135 COLNNOT01 g506882 C elegans
    SEQ ID NO:17 SEQ ID NO:66 HRM-17  641127 BRSTNOT03 g1310668 Human Hok-2 gene product
    SEQ ID NO:18 SEQ ID NO:67 HRM-18  691768 LUNGTUT02 g309183 Mus musculus
    SEQ ID NO:19 SEQ ID NO:68 HRM-19  724157 SYNOOAT01 g577542 C. elegans C16C10
    SEQ ID NO:20 SEQ ID NO:69 HRM-20  864683 BRAITUT03 g1418563 C. elegans
    SEQ ID NO:21 SEQ ID NO:70 HRM-21  933353 CERVNOT01 g1657672 C. elegans
    SEQ ID NO:22 SEQ ID NO:71 HRM-22 1404643 LATRTUT02 g459002 C. elegans
    SEQ ID NO:23 SEQ ID NO:72 HRM-23 1561587 SPLNNOT04 g868266 C. elegans
    SEQ ID NO:24 SEQ ID NO:73 HRM-24 1568361 UTRSNOT05 g1834503 Human mucin
    SEQ ID NO:25 SEQ ID NO:74 HRM-25 1572888 LNODNOT03 g603396 S. cerevisiae YER156c
    SEQ ID NO:26 SEQ ID NO:75 HRM-26 1573677 LNODNOT03 g849195 S. cerevisiae D9481.16
    SEQ ID NO:27 SEQ ID NO:76 HRM-27 1574624 LNODNOT03 g1067025 C. elegans R07E5.14
    SEQ ID NO:28 SEQ ID NO:77 HRM-28 1577239 LNODNOT03 g728657 S. cerevisiae
    SEQ ID NO:29 SEQ ID NO:78 HRM-29 1598203 BLADNOT03 g1200033 C. elegans F35G2
    SEQ ID NO:30 SEQ ID NO:79 HRM-30 1600438 BLADNOT03 g286001 KIAA0005
    SEQ ID NO:31 SEQ ID NO:80 HRM-31 1600518 BLADNOT03 g790405 C. elegans
    SEQ ID NO:32 SEQ ID NO:81 HRM-32 1602473 BLADNOT03 g1574570 Haemophilus influenzae
    SEQ ID NO:33 SEQ ID NO:82 HRM-33 1605720 LUNGNOT15 g1055080 C. elegans
    SEQ ID NO:34 SEQ ID NO:83 HRM-34 1610501 COLNTUT06 g313741 S. cerevisiae YBL0514
    SEQ ID NO:35 SEQ ID NO:84 HRM-35 1720770 BLADNOT06 g1006641 C. elegans F46C5
    SEQ ID NO:36 SEQ ID NO:85 HRM-36 1832295 BRAINON01 g561637 Human enigma protein
    SEQ ID NO:37 SEQ ID NO:86 HRM-37 1990522 CORPNOT02 g558396 S. cerevisiae
    SEQ ID NO:38 SEQ ID NO:87 HRM-38 2098087 BRAITUT02 g1066284 Mus musculus uterine mRNA
    SEQ ID NO:39 SEQ ID NO:88 HRM-39 2112230 BRAITUT03 g861306 C. elegans
    SEQ ID NO:40 SEQ ID NO:89 HRM-40 2117050 BRSTTUT02 g687821 C. elegans
    SEQ ID NO:41 SEQ ID NO:90 HRM-41 2184712 SININOT01 g868241 C. elegans C56C10
    SEQ ID NO:42 SEQ ID NO:91 HRM-42 2290475 BRAINON01 g733605 C. elegans
    SEQ ID NO:43 SEQ ID NO:92 HRM-43 2353452 LUNGNOT20 g1507666 Schizosaccharomyces pombe
    SEQ ID NO:44 SEQ ID NO:93 HRM-44 2469611 THP1NOT03 g1495332 C. elegans
    SEQ ID NO:45 SEQ ID NO:94 HRM-45 2515476 LIVRTUT04 g1665790 KIAA0262
    SEQ ID NO:46 SEQ ID NO:95 HRM-46 2754573 THP1AZS08 g478990 Human RNA binding protein
    SEQ ID NO:47 SEQ ID NO:96 HRM-47 2926777 TLYMNOT04 g687823 C. elegans
    SEQ ID NO:48 SEQ ID NO:97 HRM-48 3217567 TESTNOT07 g1841547 Human HLA class III region
    SEQ ID NO:49 SEQ ID NO:98 HRM-49 3339274 SPLNNOT10 g1177434 Human mRNA
  • HRM-1 (SEQ ID NO:1) was identified in Incyte Clone 133 from the U937NOT01 CDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:50, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 133 (U937NOT01), 013508 (THP1PLB01), 210174 (SPLNNOT02), 1655863 (PROSTUT08), 1725724 (PROSNOT14), 1858205 (PROSNOT18), and 2646014 (OVARTUT05). [0060]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO: 1. HRM-1 is 151 amino acids in length and has four potential phosphorylation sites at T2, S14, S69, and T111. HRM-1 has sequence homology with human KIAA0105 (g285947) and is found in cDNA libraries which have proliferating cells and are associated with cancer or immune response. [0061]
  • HRM-2 (SEQ ID NO:2) was identified in Incyte Clone 1762 from the U937NOT01 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:51, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 1762 (U937NOT01), 1254927 (LUNGFET03), and 2070865 (ISLTNOT01). [0062]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:2. HRM-2 is 185 amino acids in length and has a potential N glycosylation site at N108; eight potential phosphorylation sites at T22, S26, T27, S31, T51, T70, and T135; a leucine zipper motif at L[0063] 136KDVVWGLNSLFTDLLNFDDPL; and a ubiquitin conjugation motif at W105HPNITETGEICLSL. HRM-2 has sequence homology with a gene from Ascaris suum (g1518121) and is found in cDNA libraries which have secretory or proliferating cells and are associated with development.
  • HRM-3 (SEQ ID NO:3) was identified in Incyte Clone 1847 from the U937NOT01 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:52, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 274 (U937NOT01), 1847 (U937NOT01), 262233 (HNT2AGTO1), 972977 (MUSCNOT02), and 1859611(PROSNOT18). [0064]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:3. HRM-3 is 59 amino acids in length and has four potential N glycosylation sites at N147, N352, N410, and N421, and 17 potential phosphorylation sites at S13, T21, S43, S89, S131, S207, T243, S278, T286, S335, S337, S350, S354, S369, S380, S412, and S542. HRM-3 has sequence homology with a [0065] saccharomyces cerevisiae protein (g130221 1) and is found in cDNA libraries which have proliferating or immortalized cells.
  • HRM-4 (SEQ ID NO:4) was identified in Incyte Clone 9337 from the HMC1NOT01 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:53, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 9337 (HMC1NOT01), 670279 (CRBLNOT01), 717305 (PROSTUT01), 968249 (BRSTNOT05), and 1546506 (PROSTUT04). [0066]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:4. HRM-4 is 338 amino acids in length and has a potential N glycosylation site at N327, 11 potential phosphorylation sites at T15, S36, S42, S50, T51, S73, S144, S176, T256, S140, and T329; and five zinc finger motifs at C[0067] 192RC194SECGKI FRNPRYFSVHKKIH, C222QDCGKGFVQSSSLTQHQRVH, C250OQECGRTFNDRSAISQHLRTH, C278QDCGKAFRQSSHLIRHQRTH, and C306NKCGKAFTQSSHLIGHQRTH. HRM-4 has sequence homology with a human zinc finger protein (g1613852) and is found in cDNA libraries which have proliferating, cancerous, or secretory cells.
  • HRM-5 (SEQ ID NO:5) was identified in Incyte Clone 9476 from the HMCINOT01 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:54, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 9476 (HMC1NOT01), 010403 (THP1PLB01), 495099 (HNT2NOT01), 1670783 (BMARNOT03), 1997203 (BRSTTUT03), and 2190637 (THYRTUT03). [0068]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:5. HRM-5 is 456 amino acids in length and has a potential N glycosylation site at N385; 14 potential phosphorylation sites at T9, T12, S58, T74, T163, T139, S175, T211, T239, T272, S331, T367, T420, and S443, and an ATP/GTP binding motif at G[0069] 70PPGTGKT77. HRM-5 has sequence homology with a S. cerevisiae protein (g755784) and is found in cDNA libraries which have dividing, cancerous or immortalized cells and are associated with immune response.
  • HRM-6 (SEQ ID NO:6) was identified in Incyte Clone 10370 from the THP1PLB01 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:55, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 010370 (THP1PLB01), 109018 (AMLBNOT01), 259388 (HNT2RAT01), and 1518624 (BLADTUT04). [0070]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:6. HRM-6 is 210 amino acids in length and has one potential N-glycosylation site at N11 and nine potential phosphorylation sites at T13, T21, T46, T124, S125, S132, T143, T167, and T191. HRM-6 has sequence homology with a putative p64 CLCP human protein (g895845) and is found in cDNA libraries which have dividing, cancerous or immortalized cells and are associated with immune response. [0071]
  • HRM-7 (SEQ ID NO:7) was identified in Incyte Clone 30137 from the THP1PLB01 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:56, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 30137 (THP1NOB01), 531638 (BRAINOT03), 1653122 (PROSTUT08), and 1682227 (PROSNOT15). [0072]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:7. HRM-7 is 255 amino acids in length and has one potential N glycosylation site at N86 and 12 potential phosphorylation sites at T9, T28, S32, S61, S94, S142, S156, S160, T169, S118, S220, and S236. HRM-7 has sequence homology with human clone 23733 (g1710241) and is found in cDNA libraries which have dividing, cancerous or immortalized cells and are associated with immune response. [0073]
  • HRM-8 (SEQ ID NO:8) was identified in Incyte Clone 77180 from the SYNORAB01cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:57, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 077180 (SYNORAB01), 604706 (BRSTTUT01), 977901 (BRSTNOT02), 1870373 (SKINBIT01), and 2169441 (ENDCNOT03). [0074]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:8. HRM-8 is 188 amino acids in length and has one potential amidation site, Q170GKR; two potential N glycosylation sites at N60 and N68; and four potential phosphorylation sites at S70, T164, T166, and S183. HRM-8 has sequence homology with a S. [0075] cerevisiae protein (g5372) and is found in cDNA libraries which have dividing, cancerous or immortalized cells and are associated with immune response.
  • HRM-9 (SEQ ID NO:9) was identified in Incyte Clone 98974 from the PITUNOR01 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:58, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 98974 (PITUNOR01), 443924 (MPHGNOT03), 1401540 (BRAITUT08), 1507305 (BRAITUT07), 1700814 (BLADTUT05), and 1809947 (PROSTUT12). [0076]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:9. HRM-9 is 531 amino acids in length and has one potential N glycosylation site at N480; 37 potential phosphorylation sites at S19, T22, S38, T64, T76, T91, Si117, Si118, S158, T164, T177, T182, T200, T267, Y281, Y311, Y322, S333, S394, S402, S404, S409, S414, S416, S418, S429 S434, S439, S440, S456, S460, S466, S478, S505, S510, S524, S528, and one potential glycosaminoglycan motif at S434GSG. HRM-9 sequence homology with a [0077] Caenorhabditis elegans protein (g1627704) and is found in cDNA libraries which have secretory, proliferating or immune cells.
  • HRM-10 (SEQ ID NO: 10) was identified in Incyte Clone 118160 from the MUSCNOT01 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:59, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 118160 (MUSCNOT01), 323015 (EOSIHET02), and 1856519 (PROSNOT18). [0078]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:10. HRM-10 is 348 amino acids in length and has two potential N glycosylation sites at N150 and N317; 17 potential phosphorylation sites at T23, T45, S60, T126, S130, S140, S145, S151, S154, S158, S[0079] 186, Y208, Y234, S217, T271, T303, and S327, and a transcription factor signature at C310SKCKKKNCTYNQVQTRSA DEPMTTFVLCNEC. HRM-10 has sequence homology with a Mus musculus protein (g220594) and is found in cDNA libraries which have secretory or immune associations.
  • HRM-11 (SEQ ID NO:11) was identified in Incyte Clone 140516 from the TLYMNOR01 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:60, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 140516 (TLYMNOR01), 143729 (TLYMNOR01), 1346014 (PROSNOT11), and 2074866 (ISLTNOT01). [0080]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:11. HRM-11 is 393 amino acids in length and has 14 potential phosphorylation sites at S22, T33, S41, S69, T156, Y157, S166, S199, T242, T308, T324, S350, T359, S378. HRM-11 has sequence homology with a [0081] C. elegans protein (g1086723) and is found in cDNA libraries which have proliferating, secretory or immune cells.
  • HRM-12 (SEQ ID NO: 12) was identified in Incyte Clone 207452 from the SPLNNOT02 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:61, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 207452 (SPLNNOT02), 238306 (SINTNOT02), 1559492 (SPLNNOT04), and 1852567 (LUNGFET03). [0082]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:12. HRM-12 is 320 amino acids in length and one potential amidation site at E[0083] 210GKK; two potential N glycosylation sites atN12 and N314; seven potential phosphorylation sites at S34, S51, S56, Slll, T157, S198, and S318; one potential glycosaminoglycan motif, S224GAG; one immunoglobulin major histocompatibility motif, F305FCNVFH; and two mitochondrial carrier protein signatures, P35FDVIKIRF and P138VDVLRTRF. HRM-12 has sequence homology with a S. cerevisiae protein (gl314086) and is found in cDNA libraries which have secretory and proliferating cells.
  • HRM-13 (SEQ ID NO: 13) was identified in Incyte Clone 208836 from the SPLNNOT02 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:62, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 26879 (SPLNFET01), 208836 (SPLNNOT02), and 1916142 (PROSTUT04). [0084]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:13. HRM-13 is 343 amino acids in length and has one potential N glycosylation site at N172; 17 potential phosphorylation sites at S45, S46, T62, S73, S84, S85, S102, S105, T124, S137, Y153, T192, S216, Y226, Y241, S253 and T293; and a zinc finger motif at C[0085] 277RHYFCESCA. HRM-13 has sequence homology with a S. cerevisiae protein (g662126) and is found in cDNA libraries which have proliferating cells and are associated with immune response.
  • HRM-14 (SEQ ID NO:14) was identified in Incyte Clone 569710 from the MMLR3DT01 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:63, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 145344 (TLYMNOR01) and 569710 (MMLR3DT01). [0086]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:14. HRM-14 is 368 amino acids in length and has 10 potential phosphorylation sites at S5, T16, T125, S132, S142, S157, S167, S185, S208, and S246; and four zinc finger motifs at C[0087] 253DECGKHFSQGSALILHQRIH, C281,VECGKAFSRSSILVQH QRVH, C309LECGKAFSQNSGLINHQRIH, and C337VQCGKSYSQSSNLFRHQRRH. HRM-14 has sequence homology with a human zinc finger protein (gl698719) and is found in cDNA libraries which are associated with immune response.
  • HRM-15 (SEQ ID NO:15) was identified in Incyte Clone 606742 from the BRSTTUT01 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:64, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 606742 (BRSTIUT01) and 1559478 (SPLNNOT04). [0088]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:15. HRM-15 is 158 amino acids in length and has two potential myristylation sites, G92GFHGQ and G96QMHSR, and one potential PKC phosphosphorylation site, S40. HRM-15 has sequence homology with human clone 23679 (g1710201) and is found in cDNA libraries with proliferating, secretory and/or cancerous cells. [0089]
  • HRM-16 (SEQ ID NO: 16) was identified in Incyte Clone 611135 from the COLNNOT01 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:65, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 611135 (COLNNOT01), 659029 (BRAINOT03), and 1861691 (PROSNOT19). [0090]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:16. HRM-16 is 334 amino acids in length and has 11 potential phosphorylation sites at S17, T29, T128, S133, S162, S176, S263, T257, S263, S277, and S294. HRM-16 has sequence homology with a [0091] C. elegans protein (g506882) and is found in cDNA libraries with secretory cells.
  • HRM-17 (SEQ ID NO: 17) was identified in Incyte Clone 641127 from the BRSTNOT03 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:66, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 641127 (BRSTNOT03) and 673153 (CRBLNOT01). [0092]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO: 17. HRM-17 is 488 amino acids in length and has one N glycosylation site at N215; 11 potential phosphorylation sites at S70, S78, S92, T102, S111, T190, Y235, S303, S329, S415, and T471; and eight zinc finger motifs at C[0093] 237EQCGKGFTRSSSLLIHQAVH, C265DKCGKGFTRSSSLLIHHAVH, C293DKCGKGFSQSSKLHIHQRVH, C321,EECGMSFS QRSNLHIHQRVH, C349GECGKGFSQSSNLHIHRCIH, C377YECGKGFSQSSDLRIHLRVH, C405GKCGKGFSQSSKLLIHQRVH, and C433SKCGKGFSQSSNLHIHQRVH. HRM-17 has sequence homology with a human HOK-2 gene product (g1310668) and is found in cDNA libraries associated with sensory and secretory functions.
  • HRM-18 (SEQ ID NO:18) was identified in Incyte Clone 691768 from the LUNGTUT02 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:67, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 691768 (LUNGTUT02), 1417161 (BRAINOT12) and 1931861 (COLNNOT16). [0094]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:18. HRM-18 is 255 amino acids in length and has one potential N glycosylation site at N102 and 13 potential phosphorylation sites at S21, T90, T109, S111, T124, S134, S139, T141, S158, S172, S181, S187, and T206. HRM-18 has sequence homology with a [0095] M. musculus protein (g309183) and is found in cDNA libraries with proliferating or cancerous cells.
  • HRM-19 (SEQ ID NO:19) was identified in Incyte Clone 724157 from the SYNOOAT01 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:68, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 724157 (SYNOOAT01), 1516153 (PANCTUT01), and 1610152 (COLNTUT06). [0096]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO: 19. HRM-19 is 351 amino acids in length and has eight potential phosphorylation sites at T30, S41, S53, T135, S172, S187, T273, and S331; one potential glycosaminoglycan site, S[0097] 18GTG; and one potenti mitochondrial carrier motif, P13,LDVVKVRL. HRM-19 has sequence homology with C. elegans C16C10 (g577542) and is found in cDNA libraries associated with cell proliferation, cancer and immune response.
  • HRM-20 (SEQ ID NO:20) was identified in Incyte Clone 864683 from the BRAITUT03 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:69, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 486297 (HNT2RATO1), 864683 (BRAITUT03), 1314465 (BLADTUT02), 1610776 (COLNTUT06), 1856771 (PROSNOT18), 1866081 (PROSNOT19), 1932221 (COLNNOT16), and 2125225 (BRSTNOT07). [0098]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:20. HRM-20 is 535 amino acids in length and has three potential N glycosylation sites at N202, N252, and N523; and 17 potential phosphorylation sites at S2, S12, S42, S49, S102, S157, T165, T171, T232, T255, T317, S332, S428, T441, S453, S500, and S509. HRM-20 has sequence homology with a [0099] C. elegans protein (g1418563) and is found in cDNA libraries associated with cell proliferation, cancer and immune response.
    HRM-21 (SEQ ID NO:21)
  • Was identified in Incyte Clone 933353 from the CERVNOT01 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:70, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 928904 (BRAINOT04), 933353 (CERVNOT01), and 2452674 (ENDANOT01). [0100]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:21. HRM-21 is 201 amino acids in length and has one potential N glycosylation site at N82; five potential phosphorylation sites at T70, S83, S98, S154, and Ti 87; and one tyrosine phosphatase motif at V[0101] 130HCKAGRSRSATM. HRM-21 has sequence homology with a C. elegans protein (g1657672) and is found in cDNA libraries associated with immune response.
    HRM-22 (SEQ ID NO:22)
  • Was identified in Incyte Clone 1404643 from the LATRTUT02 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:71, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 878243 (LUNGAST01), 1404643 (LATRTUT02), 1508343 (LUNGNOT14) and 2585156 (BRAITUT22). [0102]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:22. HRM-22 is 239 amino acids in length and has four potential phosphorylation sites at S5, S89, S133, and T211. HRM-22 has sequence homology with a [0103] C. elegans protein (g459002) and is found in cDNA libraries associated with cell proliferation, cancer and immune response.
    HRM-23 (SEQ ID NO:23)
  • Was identified in Incyte Clone 1561587 from the SPLNNOT04 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:72, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 522573 (MMLR2DTO1), 773822 (COLNNOT05), 1304839 (PLACNOT02), 1381253 (BRAITUT08), 1452511 (PENITUT01), 1539060 (SINTIUT01), 1561587 (SPLNNOT04), and 2416572 (HNT3AZTO1). [0104]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:23. HRM-23 is 244 amino acids in length and has five potential phosphorylation sites at T40, S75, T84, T89, and S194. HRM-23 has sequence homology with a [0105] C. elegans protein (g868266) and is found in cDNA libraries associated with cell proliferation, cancer and immune response.
    TABLE 1
    Protein Nucleotide Abbreviation Clone ID Library NCBI Homolog
    SEQ ID NO:1 SEQ ID NO:50 HRM-1    133 U937NOT01 g285947 KIAA0105
    SEQ ID NO:2 SEQ ID NO:51 HRM-2    1762 U937NOT01 g1518121 Ascaris suum
    SEQ ID NO:3 SEQ ID NO:52 HRM-3    1847 U937NOT01 g1302211 Saccharomyces cerevisiae
    SEQ ID NO:4 SEQ ID NO:53 HRM-4    9337 HMC1NOT01 g1613852 Human zinc finger protein (zf2)
    SEQ ID NO:5 SEQ ID NO:54 HRM-5    9476 HMC1NOT01 g755784 S. cerevisiae
    SEQ ID NO:6 SEQ ID NO:55 HRM-6   10370 THP1PLB01 g895845 Human putative p64 CLCP protein
    SEQ ID NO:7 SEQ ID NO:56 HRM-7   30137 THP1NOB01 g1710241 Human clone 23733 mRNA
    SEQ ID NO:8 SEQ ID NO:57 HRM-8   77180 SYNORAB01 g5372 S. cerevisiae
    SEQ ID NO:9 SEQ ID NO:58 HRM-9   98974 PITUNOR01 g1627704 Caenorhabditis elegans
    SEQ ID NO:10 SEQ ID NO:59 HRM-10  118160 MUSCNOT01 g220594 Mus musculus
    SEQ ID NO:11 SEQ ID NO:60 HRM-11  140516 TLYMNOR01 g1086723 C. elegans
    SEQ ID NO:12 SEQ ID NO:61 HRM-12  207452 SPLNNOT02 g1314086 S. cerevisiae
    SEQ ID NO:13 SEQ ID NO:62 HRM-13  208836 SPLNNOT02 g662126 S. cerevisiae
    SEQ ID NO:14 SEQ ID NO:63 HRM-14  569710 MMLR3DT01 g1698719 Human zinc finger protein
    SEQ ID NO:15 SEQ ID NO:64 HRM-15  606742 BRSTTUT01 g1710201 Human clone 23679 mRNA
    SEQ ID NO:16 SEQ ID NO:65 HRM-16  611135 COLNNOT01 g506882 C elegans
    SEQ ID NO:17 SEQ ID NO:66 HRM-17  641127 BRSTNOT03 g1310668 Human Hok-2 gene product
    SEQ ID NO:18 SEQ ID NO:67 HRM-18  691768 LUNGTUT02 g309183 Mus musculus
    SEQ ID NO:19 SEQ ID NO:68 HRM-19  724157 SYNOOAT01 g577542 C. elegans C16C10
    SEQ ID NO:20 SEQ ID NO:69 HRM-20  864683 BRAITUT03 g1418563 C. elegans
    SEQ ID NO:21 SEQ ID NO:70 HRM-21  933353 CERVNOT01 g1657672 C. elegans
    SEQ ID NO:22 SEQ ID NO:71 HRM-22 1404643 LATRTUT02 g459002 C. elegans
    SEQ ID NO:23 SEQ ID NO:72 HRM-23 1561587 SPLNNOT04 g868266 C. elegans
    SEQ ID NO:24 SEQ ID NO:73 HRM-24 1568361 UTRSNOT05 g1834503 Human mucin
    SEQ ID NO:25 SEQ ID NO:74 HRM-25 1572888 LNODNOT03 g603396 S. cerevisiae YER156c
    SEQ ID NO:26 SEQ ID NO:75 HRM-26 1573677 LNODNOT03 g849195 S. cerevisiae D9481.16
    SEQ ID NO:27 SEQ ID NO:76 HRM-27 1574624 LNODNOT03 g1067025 C. elegans R07E5.14
    SEQ ID NO:28 SEQ ID NO:77 HRM-28 1577239 LNODNOT03 g728657 S. cerevisiae
    SEQ ID NO:29 SEQ ID NO:78 HRM-29 1598203 BLADNOT03 g1200033 C. elegans F35G2
    SEQ ID NO:30 SEQ ID NO:79 HRM-30 1600438 BLADNOT03 g286001 KIAA0005
    SEQ ID NO:31 SEQ ID NO:80 HRM-31 1600518 BLADNOT03 g790405 C. elegans
    SEQ ID NO:32 SEQ ID NO:81 HRM-32 1602473 BLADNOT03 g1574570 Haemophilus influenzae
    SEQ ID NO:33 SEQ ID NO:82 HRM-33 1605720 LUNGNOT15 g1055080 C. elegans
    SEQ ID NO:34 SEQ ID NO:83 HRM-34 1610501 COLNTUT06 g313741 S. cerevisiae YBL0514
    SEQ ID NO:35 SEQ ID NO:84 HRM-35 1720770 BLADNOT06 g1006641 C. elegans F46C5
    SEQ ID NO:36 SEQ ID NO:85 HRM-36 1832295 BRAINON01 g561637 Human enigma protein
    SEQ ID NO:37 SEQ ID NO:86 HRM-37 1990522 CORPNOT02 g558396 S. cerevisiae
    SEQ ID NO:38 SEQ ID NO:87 HRM-38 2098087 BRAITUT02 g1066284 Mus musculus uterine mRNA
    SEQ ID NO:39 SEQ ID NO:88 HRM-39 2112230 BRAITUT03 g861306 C. elegans
    SEQ ID NO:40 SEQ ID NO:89 HRM-40 2117050 BRSTTUT02 g687821 C. elegans
    SEQ ID NO:41 SEQ ID NO:90 HRM-41 2184712 SININOT01 g868241 C.elegans C56C10
    SEQ ID NO:42 SEQ ID NO:91 HRM-42 2290475 BRAINON01 g733605 C. elegans
    SEQ ID NO:43 SEQ ID NO:92 HRM-43 2353452 LUNGNOT20 g1507666 Schizosaccharomyces pombe
    SEQ ID NO:44 SEQ ID NO:93 HRM-44 2469611 THP1NOT03 g1495332 C. elegans
    SEQ ID NO:45 SEQ ID NO:94 HRM-45 2515476 LIVRTUT04 g1665790 KIAA0262
    SEQ ID NO:46 SEQ ID NO:95 HRM-46 2754573 THP1AZS08 g478990 Human RNA binding protein
    SEQ ID NO:47 SEQ ID NO:96 HRM-47 2926777 TLYMNOT04 g687823 C. elegans
    SEQ ID NO:48 SEQ ID NO:97 HRM-48 3217567 TESTNOT07 g1841547 Human HLA class III region
    SEQ ID NO:49 SEQ ID NO:98 HRM-49 3339274 SPLNNOT10 g1177434 Human mRNA
  • HRM-24 (SEQ ID NO:24) was identified in Incete Clone 1568361 from the UTRSNOT05 cDNA library using a computer search for amino acid sequence. A consensus sequence, SEQ ID NO;73, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 927874 (BRAINOT04), 1255220 (MENITUT03), 1242340 (LUNGNOT03), 13495 (LATRTU02), 1381263 (BRAITUT08), 1500028 (SINTBST01), 1568361 (UTRSNOT05), 1653237 (PROSTUT08), 1975340 (PANCTUT02), and 3274608 (PROSBPT06). [0106]
  • In one embodiment, the invention encompasses a protein compraising the amino acid sequence of SEQ ID NO:24, HRM-24 is 431 amino acids in length and has five potential N glycosylation sites at N75, N95 N171, N202, and N298; eight potential phosphorylation sites at S2, S3, T11, T13, S17, Y316, T375, and T415, and a leucine zipper motif, L[0107] 96SAFNNILSNLGYILLGLLFLL. HRM-24 has sequence homology with human mucin (g1834503) and is found cDNA libraries proliferating, cancerus or inflamed cells.
  • HRM-25 (SEQ ID NO;25) was identified in Incyte Clone 1572888 from the LNODNOT03 cDNA library using a computer search for amino acid sequence aligments. A consensus sequence SEQ ID NO:74 was derived from the extended and overlapping nucleic acid sequence: Incyte Clones 1438142 (PANCNT08), 1572888 (LNODNOT03), and 1665075 (BRSTNOT09). [0108]
  • In one embodiment, the invention encompases a protein compraising the amino acid sequence of SEQ ID NO:25. HRM-25 is 376 amino acids in length and has one N glycosylation site five potential phosphorylation sites at S111, T150, S151, T159, and, S196. HRM-25 has sequence homology with [0109] S. cerevisiae YER156c (g603396) and is found in cDNA libraries with secretory cells.
  • HRM-26 (SEQ ID NO:26) was identifie in Incyte Clone 1573677 from the LONDNOT03 cDNA library using a computer search for amino acid sequence aligments. A concensus sequence, SEQ ID NO:75, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 040360 (TBLYNOT01), 065573 (PLACNOB01), 228382 (PANCNOT01), 1457788 (COLNFET02), 1573677 (LNODNOT03), and 1854560 (HNT3AZT01) [0110]
  • In one embodiment, the invention encompasses a protein compraising the amino acid sequence of SEQ ID NO:26 is 340 amino acids in length and has one potential N glycosylation site at N213 and 13 potential phosphorylation site at T10, S22, T53, T56, S160, S168, S170, S177, S201, S226 S297, S303, and T329. HRM-26 has sequence homology with [0111] S. cerevisiae D9481.16 (g849195) and its found in cNDA libraries associated with secretion, immune response, and cancer.
  • HRM-27 (SEQ ID NO:27) was identified in Icyte Clone 1574624 from the LNODNOT03 cDNA library using a coomputer search for amino acid sequence aligments. A concesnsus sequence, SEQ ID NO:76 was derived from the extended and overlapping nucleic acid sequence: Incyte Clones 90012 (HYPONOB01), 888491 (STOMTUT01), and 1574624 (LNODNOT03). [0112]
  • In one embodimen, the invention encompasses a protein comprising the amino acids sequence of SEQ ID NO:27. HRM-27 is 174 amino acids in length and has one N glycosylation site at N51 and five potential phosphorylation sites at S111, T150, S151, T159, and T196. HRM-27 has sequence homology with a [0113] C. elegants protein (g1067025) and is found in cDNA libraries associated with secretion, immune responce and ancer.
  • HRM-28 (SEQ ID NO:28) was identified in Incyte Clones 1577239 from the LNODNOT03 cDNA library using a computer search for amino acid sequence aligments. A concensus sequence, SEQ ID NO:77, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 100565 (ADRENOT01), 1336693 (COLNNOT13 ), and 1577239 (LNODNOT03). [0114]
  • In one embodiment, the invention encompasses a protein comprising the amino acids sequence of SEQ ID NO:28, HRM-28 is 179 amino acids in length and has one potential N glycosylation site at N60 and five potential phosphorylation site at Y61, S62, Y104, T136, and Y142. HRM-28 has sequence homology with a [0115] S. cerevisiae protein (g728657) and is found in cDNA libraries associated with sevretion and immune response.
  • HRM-29 (SEQ ID NO:29) was identified in Incyte Clone 1598203 from the BLADNOT-03 cDNA library using a computer search for amino acid sequence aligments. A consensus sequence, SEQ ID NO:78, was derived from the extended and overlapping nucleic acid sequences: Incyte Clone 1598203 (BLADNOT03), 1697035 (COLNNOT23), and 1932332 (COLNNOT16). [0116]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO;29. HRM-29 is 205 amino acids in length and has one potential N glycocylation site at N117 and five potential phosphorylation sites at T68, T118, S137, S140, and S159. HRM-29 has sequence homology with a [0117] C. elegants protein (g1200033) and is found in cDNA libraries associated with secretion.
  • HRM-30 (SEQ ID NO:30) was identified in Incyte Clone 1600438 fron the BLADNOT03 cDNA library using a computer search for amino acid sequence aligments. A consensus sequence, SEQ ID NO:79, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 835283 (PROSNOT07), 1600044 (BLADNOT03), 1600438 (BLANDOT03), and 1922072 (BRSTTUT01). [0118]
  • In one embodiment, the invention encompasses a protein comprising the acid sequenceof SEQ ID NO:30. HRM-30 is 419 amino acids in length and has one potential N glycosylation site at N161; twelve potential phosphorylation sites at T16, S57, T67, T83, S100, T107, S144, S206, T254, Y351, S412, and S414; a leucine zipper motif, l[0119] 38NEAGDDLEAVAKFLDSGSRL; and an ATP/GTP binding motif, A385HVAKGKS. HRM-30 has sequence homology with human KIAA0005 (g286001) and is found in cDna libraries associated with secretion and cancer.
  • HRM-31 (SEQ ID NO:31) was identified in Incyte Clone 1600518 from the BLADNOT03 cNDA library using a computer search for amino acid sequence aligment. A consensus sequence, SEQ ID NO;80, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 389679 (THYMNOT02), 1600518 (BLADNOT03), 2055734 (BEPINOT02), and 2509270 CONUTUT01). [0120]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:31. HRM-31 is 376 amino acid in length and has one potential N glycosylation site at N161 and 14 potential phosphorylation sites at T30, S65, S75, S95, S106, T134, S159, S224, T228, T250, T292, S299, T303, and S323 and a glycosaminoglycan motif, S14 GPG. HRM-31 has sequence homology with a [0121] C. elegants protein (g790405) and is found in cNDA libraries associated with immune response, secretion and cancer.
  • HRM-32 (SEQ ID NO:32) was identified in Incyte Clone 1602473 from the BLADNOT03 cDNA library using a computer search for amino acid sequence aligments. A consensus sequence, SEQ ID NO:81, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 1351857 (LATRTUT02), 1602473 (BLADNOT03), and 2478778 (SMCANOT01). [0122]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:32. HRM-32 is 237 amino acids in length and has seven potential phosphorylation site at T51, T68, S92, S143, T171, S193, and S203. HRM-32 has sequence homology with a [0123] Haemophilus influenzae protein (g1574570) and is found in cDNA libraries associated with immune response, and cancer.
  • HRM-33 (SEQ ID NO:33) was identified in Incyte Clone 16057220 from the LUNGNOT15 cDNA library using a computer search for amino acid sequence aligment. A consensus sequence, SEQ ID NO:82, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 660915 (BRAINOT03), 1347135 (PROSNOT11), and 1605720 (LUNGNOT15). [0124]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:33. HRM-33 is 152 amino acids in length and has four potential phosphorylation sites at S10, S23, T34, and S66; and a leucine zipper motif, L[0125] 77AVGNYRLKEYEKALKYVRGLL. HRM-33 has sequence homology with C. elegans (g155080) and is found in cDNA libraries associated with secretion and immune response.
  • HRM-34 (SEQ ID NO:34) was identified in Incyte Clone 1610501 from the COLNTUT06 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:83, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 1610501 (COLNTUT06) and 2477716 (SMCANOT01). [0126]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:34. HRM-34 is 179 amino acids in length and has five potential phosphorylation sites at S32, S48, T45, T50, and T52. HRM-34 has sequence homology with a [0127] S. cerevisiae protein (g313741) and is found in cDNA libraries associated with cancer and immune response.
  • HRM-35 (SEQ ID NO:35) was identified in Incyte Clone 1720770 from the BLADNOT06 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:84, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 681455 (UTRSNOT02), 813292 (LUNGNOT04), 1223029 (COLNTUT02), 1444186 (THYRNOT03), 1522592 (BLADTUT04), 1720770 (BLADNOT06), and 1798409 (COLNNOT27). [0128]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:35. HRM-35 is 196 amino acids in length and has an amidation motif, H[0129] 179GKR, and seven potential phosphorylation sites at S2, S6, S31,S84, S90, T136, and T161. HRM-35 has sequence homology with a C.elegans protein (g1006641) and is found in cDNA libraries associated with secretion, immune response, and cancer.
  • HRM-36 (SEQ ID NO:36) was identified in Incyte Clone 1832295 from the BRAINON01 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:85, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 060275 (LUNGNOT01), 1823989 (GBLATUT01), and 1832295 (BRAINON01). [0130]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:36. HRM-36 is 612 amino acids in length and has 12 potential N glycosylation sites at N36, N95, N139, N146, N151, N176, N188, N226, N243, N353, N371, and N482; and 16 potent at S58, S92, S112, T153, T198, T248, S308, S373, T400, T420, T428, Y438, T458, T472, S527, and S556. HRM-36 has sequence homology with human enigma protein (g561[0131] 637) and is found in cDNA libraries associated with secretion and immune response.
  • HRM-37 (SEQ ID NO:37) was identified in Incyte Clone 1990522 from the CORPNOT02 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:86, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 264363 (HNT2AGTO0), 1990522 (CORPNOT02), and 2451448 (ENDANOT01). [0132]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:37. HRM-37 is 101 amino acids in length and has a PKC phosphorylation site at S62. HRM-37 has sequence homology with a [0133] S. cerevisiae protein (g558396) and is found in cDNA libraries associated with immune response.
  • HRM-38 (SEQ ID NO:38) was identified in Incyte Clone 2098087 from the BRAITUT02 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:87, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 690359 (LUNGTUT02), 1429907 (SINTBST01), and 2098087 (BRAITUT02). [0134]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:38. HRM-38 is 132 amino acids in length and has a potential ATP/GTP binding motif at G[0135] 74ARNLLKS. HRM-38 has sequence homology with M. musculus uterine protein (g166284) and is found in cDNA libraries associated with immune response.
  • HRM-39 (SEQ ID NO:39) was identified in Incyte Clone 2112230 from the BRAITUT03 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:88, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 1383278 (BRAITUT08), 1646103 (PROSTUT09), 2112230 (BRAITUT03), and 2510591 (CONUTUT01). [0136]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:39. HRM-39 is 188 amino acids in length and has a potential N glycosylation site at N87 and eight potential phosphorylation sites at T10, T28, S74, S93, T121, T128, Y168, and T169. HRM-39 homology with a [0137] C. elegans protein (g861306) and is found in cDNA libraries from cancerous tissues.
  • HRM-40 (SEQ ID NO:40) was identified in Incyte Clone 2117050 from the BRAITUT02 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:89, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 941515 (ADRENOT03), 1549443 (PROSNOT06), 2113261 (BRAITUT03), 2117050 (BRSTTUT02), and 2530536 (GBLANOT02). [0138]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:40. HRM-40 is 86 amino acids in length and has a potential N glycosylation site at N58 and four potential phosphorylation sites at T2, S9, T26, and T27. HRM-40 has sequence homology with a [0139] C. elegans protein (g687821) and is found in cDNA libraries involved in cell proliferation, secretion, cancer, and immune response.
  • HRM-41 (SEQ ID NO:41) was identified in Incyte Clone 2184712 from the SININOT01 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:90, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 922736 (RATRNOT02), 1976003 (PANCTUT02), and 2184712 (SININOT01). [0140]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:41. HRM-41 is 222 amino acids in length and has a potential amidation site, K[0141] 10GKK; a potential glycosaminoglycan site, S2GLG; a potential N glycosylation site, N95; and seven potential phosphorylation sites at T18, T29, T50, S84, T98, S112, and S188. HRM-41 has sequence homology with a C. elegant protein (g868241) and is found in cDNA libraries involved in cell proliferation, secretion, cancer, and immune response.
  • HRM-42 (SEQ ID NO:42) was identified in Incyte Clone 2290475 from the BRAINON01 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:91, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 238339 (SINTNOT02), 1657945 (URETTUT01), 1848691 (LUNGFET03), 2044604 (THPlT7T01), 2290475 (BRAINON01), and 2514944 (LIVRTUT04). [0142]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:42. HRM-42 is 300 amino acids in length and has a potential N glycosylation site, N5; seven potential phosphorylation sites at S23, S71, S132, S142, T176, T192, and S293; and a Mutt signature, G[0143] 165MVDPGEKISATLKREFGEE. HRM-42 has sequence homology with a C. elegans protein (g733605) and is found in cDNA libraries involved in cell proliferation, secretion, cancer, and immune response.
  • HRM-43 (SEQ ID NO:43) was identified in Incyte Clone 2353452 from the LUNGNOT20 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:92, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 1000164 (BRSTNOT03), 1308080 (COLNFET02), 1900151 (BLADTUT06), and 2353452 (LUNGNOT20). [0144]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:43. HRM-43 is 112 amino acids in length and has six potential phosphorylation sites at T23, T43, S44, T79, T84, and T98. HRM-43 has sequence homology with a [0145] Schizosaccharomvces pombe protein (gl507666) and is found in cDNA libraries involved in cell proliferation, secretion, cancer, and immune response.
  • HRM-44 (SEQ ID NO:44) was identified in Incyte Clone 2469611 from the THPlNOT03 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:93, was derived from the extended and overlapping nucleic acid sequences: Incyte clones 003088 (HMClNOT01), 1448981 (PLACNOT02), 1453563 (PENITUT01), 1824146 (GBLATUT01), 2369282 (ADRENOT07), 2469611 (THPlNOT03), and 2622587 (KERANOT02). [0146]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:44. HRM-44 is 251 amino acids in length and has a potential glycosaminoglycan site, S218GFG, and four potential phosphorylation sites at T8, S83, S212, and S226. HRM-44 has sequence homology with a [0147] C. elegans protein (gl495332) and is found in cDNA libraries involved in cell proliferation, secretion, cancer, and immune response.
  • HRM-45 (SEQ ID NO:45) was identified in Incyte Clone 2515476 from the LIVRTUT04 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:94, was derived from the extended and overlapping nucleic acid sequences: Incyte clones 18414 (HUVELPB01), 78341 (SYNORAB01), 143277 (TLYMNOR01), 181574 (PLACNOB01), 832996 (PROSTUT04), 962753 (BRSTTUT03), 1413604 (BRAINOT12), and 2515476 (LIVRTUT04). [0148]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:45. HRM-45 is 811 amino acids in length and has three potential amidation sites at G[0149] 113GRR, W165GKR, and G790GKK; four potential N glycosylation sites at N22, N56, N79, and N145; 24 potential phosphorylation sites at T11, S13, S30, S60, Y71, S81, S85, S86, S103, S254, S256, T377, S388, S425, S456, S487, T544, S552, S574, T659, S678, S702, S746, and S753; a potential glycosaminoglycan site, S160GHG; and a potential zinc finger motif at C240GHIFCWACI. HRM-45 has sequence homology with human KIAA0262 (g1665790) and is found in cDNA libraries involved in cell proliferation, secretion, cancer, and immune response.
  • HRM-46 (SEQ ID NO:46) was identified in Incyte Clone 2754573 from the THPlAZSO8 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:95, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 263630 (HNT2AGTO1), 412307 (BRSTNOT01), 491644 (HNT2AGTO1), 1253094 (LUNGFET03), 2270603 (PROSNON01), 2280508 (PROSNON01), 2375670 (ISLTNOT01), 2754573 (THPlAZS08), and 3151587 (ADRENON04). [0150]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:46. HRM-46 is 352 amino acids in length and has two potential N glycosylation sites at N141 and N294, and thirteen potential phosphorylation sites at S8, T67, T106, T110, T121, S122, S169, S206, T210, S215, S256, S260, and T296. HRM-46 has sequence homology with human RNA binding protein (g478990) and is found in cDNA libraries involved in cell proliferation, secretion, and immune response. [0151]
  • HRM-47 (SEQ ID NO:47) was identified in Incyte Clone 2926777 from the TLYMNOT04 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:96, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 040208 (TBLYNOT01), 900242 (BRSTTUT03), 963500 (BRSTTUT03), 1996474 (BRSTTUT03), and 2926777 (TLYMNOT04). [0152]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:47. HRM-47 is 432 amino acids in length and has a potential N glycosylation site at N417 and 24 potential phosphorylation sites at T51, S73, T122, T133, S177, S206, T226, T238, S293, S300, S304, S309, T325, S333, S339, S353, S360, Y361, S384, S390, T403, T412, T419, and S425 homology with a [0153] C. elegans protein (g687823) and is found in cDNA libraries involved in cell proliferation, secretion, cancer, and immune response.
  • HRM-48 (SEQ ID NO:48) was identified in Incyte Clone 3217567 from the TESTNOT07 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:97, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 905037 (COLNNOT07), 1287503 (BRAINOT11), and 3217567 (TESTNOT07). [0154]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:48. HRM-48 is 180 amino acids in length and has a potential zinc finger motif, C42GHLYCWPCL, and five potential phosphorylation sites at T33, T57, S84, T148, and S160. HRM-48 has sequence homology with human HLA class III region (g1841547) and is found in cDNA libraries involved in secretion and immune response. [0155]
  • HRM-49 (SEQ ID NO:49) was identified in Incyte Clone 3339274 from the SPLNNOT10cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:98, was derived from the extended and overlapping nucleic acid sequences: Incyte Clones 532254 (BRAINOT03), 941336 (ADRENOT03), 2447649 (THPlNOT03), and 3339274 (SPLNNOT10). [0156]
  • In one embodiment, the invention encompasses a protein comprising the amino acid sequence of SEQ ID NO:49. HRM-49 is 137 amino acids in length and has three potential phosphorylation sites at Ti 11, T91, and S119. HRM-49 has sequence homology with a deduced human translational inhibitor (g1177434) and is found in cDNA libraries involved in secretion and immune response. [0157]
  • The invention also encompasses HRM variants which retain the biological or functional activity of HRM. A preferred HRM variant is one having at least 60% amino acid sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-49. [0158]
  • The invention also encompasses polynucleotides which encode HRM. Accordingly, any nucleic acid sequence which encodes the amino acid sequence of HRM can be used to produce recombinant molecules which express HRM. In a particular embodiment, the invention encompasses a polynucleotide comprising a nucleic acid sequence selected from SEQ ID NOs:50-98 and fragment and complements thereof. [0159]
  • It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotides encoding HRM, some bearing minimal homology to the polynucleotides of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide of naturally occurring HRM, and all such variations are to be considered as being specifically disclosed. [0160]
  • Although polynucleotides which encode HRM and its variants are preferably capable of hybridizing to the polynucleotide of the naturally occurring HRM under selected conditions of stringency, it may be advantageous to produce polynucleotides encoding HRM or its derivatives possessing a different codon usage. 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. Other reasons for altering the polynucleotide encoding HRM and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence. [0161]
  • The invention also encompasses production of polynucleotides, or fragments thereof, which encode HRM and its derivatives, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents that are well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding HRM or any fragment thereof. [0162]
  • Also encompassed by the invention are polynucleotides that are capable of hybridizing to the nucleic acids of a sample, and in particular, the polynucleotides or the complements thereof shown in SEQ ID NOs:50-98, under various conditions of stringency as taught in Wahl and Berger (1987; Methods Enzymol 152:399-407) and Kimmel (1987; Methods Enzymol 152:507-511). [0163]
  • Methods for DNA sequencing which are well known and generally available in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE, Taq DNA polymerase and thermostable T7 DNA polymerase (Amersham Pharmacia Biotech (APB), Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD). Preferably, the process is automated with machines such as the MICROLAB system (Hamilton, Reno NV), DNA ENGINE thermal cycler (MJ Research, Watertown MA), and the Catalyst preparation and 373 and 377 PRISM DNA sequencing systems (ABI). [0164]
  • The nucleic acid sequences encoding HRM may be extended utilizing a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, one method which may be employed, “restriction-site” PCR, uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar (1993) PCR Methods Applic 2:318-322). In particular, genomic DNA is first amplified in the presence of primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an RNA polymerase and sequenced using reverse transcriptase. [0165]
  • Inverse PCR may also be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al. (1988) Nucleic Acids Res 16:8186). The primers may be designed using commercially available software such as OLIGO software (Molecular Insights, Cascade CO), or another program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72 C. The method uses several restriction enzymes to generate a fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template. [0166]
  • Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al. (1991) PCR Methods Applic 1:111-119). In this method, multiple restriction enzyme digestions and ligations may also be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR. [0167]
  • Another method which may be used to retrieve unknown sequences is that of Parker et al. (1991; Nucleic Acids Res 19:3055-3060). One may also use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This process avoids the need to screen libraries of cDNAs for longer sequences and is very useful in finding intron/exon junctions. [0168]
  • When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Also, random-primed libraries are preferable, in that they will contain more sequences which contain the 5′ regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5′ non-transcribed regulatory regions. [0169]
  • Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera. Output/light intensity may be converted to electrical signal using software integral to the system, 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 the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample. [0170]
  • In another embodiment of the invention, polynucleotides or fragments thereof which encode HRM may be used in recombinant DNA molecules to direct expression of HRM, portions or functional equivalents thereof, in host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode the same or a functionally equivalent amino acid sequence may be produced, and these sequences may be used to clone and express HRM. [0171]
  • As will be understood by those of skill in the art, it may be advantageous to produce HRM-encoding polynucleotides possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence. [0172]
  • The polynucleotides of the present invention can be engineered using methods generally known in the art in order to alter HRM encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the polynucleotides. For example, site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth. [0173]
  • In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding HRM may be ligated to a heterologous sequence to encode a fusion protein. For example, to screen peptide libraries for inhibitors of HRM activity, it may be useful to encode a chimeric HRM protein that can be recognized by a commercially available antibody. A fusion protein may also be engineered to contain a a cleavage site located between the HRM encoding sequence and the heterologous protein sequence, so that HRM may be cleaved and purified away from the heterologous moiety. [0174]
  • In another embodiment, sequences encoding HRM may be synthesized, in whole or in part, using chemical methods well known in the art (Caruthers et al. (1980) Nucleic Acids Symp Ser. (7) 215-223, Horn et al. (1980) Nucleic Acids Symp. Ser. (7) 225-232). Alternatively, the protein itself may be produced using chemical methods to synthesize the amino acid sequence of HRM, or a portion thereof. For example, peptide synthesis can be performed using various solid-phase techniques (Roberge et al. (1995) Science 269:202-204) and automated synthesis may be achieved, for example, using the 43 1A Peptide synthesizer (ABI). [0175]
  • The newly synthesized peptide may be purified by preparative high performance liquid chromatography (see Creighton (1983) [0176] Proteins Structures and Molecular Principles, WH Freeman, New York N.Y.). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra). Additionally, the amino acid sequence of HRM, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant protein.
  • In order to express a biologically active HRM, the polynucleotides encoding HRM or functional equivalents, may be inserted into expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. [0177]
  • Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding HRM and transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al. (1989; [0178] Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.) and Ausubel et al. (1989; Current Protocols in Molecular Biology, John Wiley & Sons, New York N.Y.).
  • A variety of expression vector/host systems may be utilized to contain and express sequences encoding HRM. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with baculovirus expression vectors; plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) or with bacterial expression vectors (Ti or pBR322 plasmids); or animal cell systems. The invention is not limited by the host cell employed. [0179]
  • The “control elements” or “regulatory sequences” are those non-translated regions of the vector enhancers, promoters, 5′ and 3′ untranslated regions--which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla CA) or the pSport1 plasmid (Life Technologies) may be used. The baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO; and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) may be cloned into the vector if the protein is to be produced in plant cells. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding HRM, vectors based on SV40 or EBV may be used with an selectable marker. [0180]
  • In bacterial systems, a number of expression vectors may be selected depending upon the use intended for HRM. For example, when large quantities of HRM are needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be used. Such vectors include, but are not limited to, the multifunctional [0181] E. coli cloning and expression vectors such as BLUESCRIPT phagemid (Stratagene), in which the sequence encoding HRM may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke and Schuster (1989) J Biol Chem 264:5503-5509); and the like. pGEX vectors (APB) may also be used to express foreign proteins as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned protein of interest can be released from the GST moiety at will.
  • In the yeast, [0182] Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel (supra) and Grant et al. (1987; Methods Enzymol 153:516-544).
  • In cases where plant cell expression is desired, the expression of sequences encoding HRM may be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu (1987) EMBO J 6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi et al. (1984) EMBO J 3:1671-1680; Broglie et al. (1984) Science 224:838-843; and Winter et al. (1991) Results Probl Cell Differ 17:85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, for example, Hobbs or Murry, hi: McGraw Hill [0183] Yearbook of Science and Technolog (1992) McGraw Hill, New York N.Y.; pp. 191-196).
  • An insect system may also be used to express HRM. For example, in one such system, [0184] Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The sequences encoding HRM may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of HRM will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses may then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which HRM may be expressed (Engelhard et al. (1994) Proc Nat Acad Sci 91:3224-3227).
  • In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding HRM may be ligated into an adenovirus transcription/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 a viable virus which is capable of expressing HRM in infected host cells (Logan and Shenk (1984) Proc Natl Acad Sci 81:3655-3659). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. [0185]
  • Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid. HACs of 6 to 10 M are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. [0186]
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding HRM. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding HRM, its initiation codon, and upstream sequences are inserted into the expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are for the particular cell system which is used, such as those described in the literature (Scharf et al. (1994) Results Probl Cell Differ 20:125-162). [0187]
  • In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the protein include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a “prepro” form of the protein may also be used to facilitate correct insertion, folding and/or function. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the ATCC (Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein. [0188]
  • For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express HRM may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques to the cell type. [0189]
  • 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 (Wigler et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy et al. (1980) Cell 22:817-23) genes which can be employed in tk- or aprt-cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler et al. (1980) Proc Natl Acad Sci 77:3567-70); npt, which confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al (1981) J Mol Biol 150:1-14); and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman and Mulligan (1988) Proc Natl Acad Sci 85:8047-51). Recently, the use of visible markers has gained popularity with such markers as anthocyanins, β glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al. (1995) Methods Mol Biol 55:121-131). [0190]
  • Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed. For example, if the sequence encoding HRM is inserted within a marker gene sequence, transformed cells containing sequences encoding HRM can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding HRM 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. [0191]
  • Alternatively, host cells which contain the nucleic acid sequence encoding HRM and express HRM 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 hybridizations 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. [0192]
  • The presence of polynucleotides encoding HRM can be detected by DNA-DNA or DNA-RNA hybridization or PCR amplification. Nucleic acid amplification based assays involve the use of oligonucleotides based on the polynucleotides encoding HRM to detect transformants containing DNA or RNA encoding HRM. [0193]
  • A variety of protocols for detecting and measuring the expression of HRM, using either polyclonal or monoclonal antibodies specific for the protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on HRM is preferred, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton et al. (1990; [0194] Serological Methods, a Laboratory Manual, APS Press, St Paul Minn.) and Maddox et al. (1983; J Exp Med 158:1211-1216).
  • A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding HRM include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding HRM, or any fragments thereof may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits (APB; Promega, Madison WI). [0195]
  • Host cells transformed with polynucleotides encoding HRM may be cultured under conditions for the expression and recovery of the protein from cell culture. The protein produced by a transformed cell may be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode HRM may be designed to contain signal sequences which direct secretion of HRM through a prokaryotic or eukaryotic cell membrane. Other constructions may be used to join sequences encoding HRM to polynucleotide encoding a protein domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex, Seattle WA). The inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego CA) between the purification domain and HRM may be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing HRM and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMAC (immobilized metal ion affinity chromatography) as described in Porath et al. (1992, Prot Exp Purif 3:263-281) while the enterokinase cleavage site provides a means for purifying HRM from the fusion protein. A discussion of vectors which contain fusion proteins is provided in Kroll et al. (1993; DNA Cell Biol 12:441-453). [0196]
  • In addition to recombinant production, portions of HRM may be produced by direct peptide synthesis using solid-phase techniques (Merrifield (1963) J Am Chem Soc 85:2149-2154). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using 431A Peptide synthesizer (ABI). Various portions of HRM may be chemically synthesized separately and combined using chemical methods to produce the full length molecule. [0197]
  • Therapeutics [0198]
  • Chemical and structural homology exits among the human regulatory proteins of the invention. The expression of HRM is closely associated with cell proliferation. Therefore, in cancers or immune disorders where HRM is an activator, transcription factor, or enhancer, and is promoting cell proliferation; it is desirable to decrease the expression of HRM. In cancers where HRM is an inhibitor or suppressor and is controlling or decreasing cell proliferation, it is desirable to provide the protein or to increase the expression of HRM. [0199]
  • In one embodiment, where HRM is an inhibitor, HRM or a portion or derivative thereof may be administered to a subject to treat a cancer such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma. Such cancers include, but are not limited to, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus. [0200]
  • In another embodiment, an agonist which is specific for HRM may be administered to a subject to treat a cancer including, but not limited to, those cancers listed above. [0201]
  • In another further embodiment, a vector capable of expressing HRM, or a portion or a derivative thereof, may be administered to a subject to treat a cancer including, but not limited to, those cancers listed above. [0202]
  • In a further embodiment where HRM is promoting cell proliferation, antagonists which decrease the expression or activity of HRM may be administered to a subject to treat a cancer such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma. Such cancers include, but are not limited to, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus. In one aspect, antibodies which specifically bind HRM may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express HRM. [0203]
  • In another embodiment, a vector expressing the complement of the polynucleotide encoding HRM may be administered to a subject to treat a cancer including, but not limited to, those cancers listed above. [0204]
  • In yet another embodiment where HRM is promoting leukocyte activity or proliferation, antagonists which decrease the activity of HRM may be administered to a subject to treat an immune response. Such responses may be associated with AIDS, Addison's disease, adult respiratory distress syndrome, allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitus, Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, atrophic gastritis, glomerulonephritis, gout, Graves' disease, hypereosinophilia, irritable bowel syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis, scleroderma, Sjbgren's syndrome, and autoimmune thyroiditis, complications of cancer, hemodialysis, extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma. In one aspect, antibodies which specifically bind HRM may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express HRM. [0205]
  • In another embodiment, a vector expressing the complement of the polynucleotide encoding HRM may be administered to a subject to treat an immune response including, but not limited to, those listed above In one further embodiment, HRM or a portion or derivative thereof may be added to cells to stimulate cell proliferation. In particular, HRM may be added to a cell in culture or cells in vivo using delivery mechanisms such as liposomes, viral based vectors, or electroinjection for the purpose of promoting cell proliferation and tissue or organ regeneration. Specifically, HRM may be added to a cell, cell line, tissue or organ culture in vitro or ex vivo to stimulate cell proliferation for use in heterologous or autologous transplantation. In some cases, the cell will have been preselected for its ability to fight an infection or a cancer or to correct a genetic defect in α disease such as sickle cell anemia, , thalassemia, cystic fibrosis, or Huntington's chorea. [0206]
  • In another embodiment, an agonist which is specific for HRM may be administered to a cell to stimulate cell proliferation, as described above. [0207]
  • In another embodiment, a vector capable of expressing HRM, or a portion or a derivative thereof, may be administered to a cell to stimulate cell proliferation, as described above. [0208]
  • In other embodiments, any of the therapeutic proteins, antagonists, antibodies, agonists, complementary sequences or vectors of the invention may be administered in combination with other therapeutic agents. Selection of the 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 of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects. [0209]
  • Antagonists or inhibitors of HRM may be produced using methods which are generally known in the art. In particular, purified HRM may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind HRM. [0210]
  • Antibodies to HRM may be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies, (i.e., those which inhibit dimer formation) are especially preferred for therapeutic use. [0211]
  • For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others, may be immunized by injection with HRM or any portion or oligopeptide thereof which has immunogenic properties. Depending on the host species, 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, keyhole limpet hemocyanin, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and [0212] Corynebacterium parvum are especially preferable.
  • It is preferred that the oligopeptides, peptides, or portions used to induce antibodies to HRM have an amino acid sequence consisting of at least five amino acids and more preferably at least 10 amino acids. It is also preferable that they are identical to a portion of the amino acid sequence of the natural protein, and they may contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of HRM amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule. [0213]
  • Monoclonal antibodies to HRM 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 hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al. (1975) Nature 256:495-497 Kozbor et al. (1985) J Immunol Methods 81:31-42 Cote et al. (1983) Pr Natl Acad Sci 80:2026-2030, Cole et al. (1984) Mol Cell Biol 62:109-120). [0214]
  • In addition, techniques developed for the production of “chimeric antibodies”, the splicing of mouse antibody genes to human antibody genes to obtain a molecule with antigen specificity and biological activity can be used (Morrison et al. (1984) Proc Natl Acad Sci 81:6851-6855, Neuberger et al. (1984) Nature 312:604-608, and Takeda et al. (1985) Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce HRM-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton (1991) Proc Natl Acad Sci 88:11120-3). [0215]
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al. (1989) Proc Natl Acad Sci 86:3833-3837, Winter et al. (1991) Nature 349:293-299). [0216]
  • Antibody fragments which contain specific binding sites for HRM may also be generated. For example, such fragments include, but are not limited to, the F(ab′)2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al. (1989) Science 254:1275-1281). [0217]
  • Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between HRM and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering HRM epitopes is preferred, but a competitive binding assay may also be employed (Maddox, supra). [0218]
  • In another embodiment of the invention, the polynucleotides encoding HRM, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, the complement of the polynucleotide encoding HRM may be used in situations in which it would be desirable to block the transcription of the mRNA. In particular, cells may be transformed with sequences complementary to polynucleotides encoding HRM. Thus, complementary molecules or fragments may be used to modulate HRM activity, or to achieve regulation of gene function. Such technology is now well known in the art, and sense or antisense oligonucleotides or larger fragments, can be designed from various locations along the coding or control regions of sequences encoding HRM. [0219]
  • Expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of polynucleotides to the targeted organ, tissue or cell population. Methods which are well known to those skilled in the art can be used to construct vectors which will express nucleic acid sequence which is complementary to the polynucleotides of the gene encoding HRM. These techniques are described both in Sambrook (ura and in Ausubel (supra). [0220]
  • Genes encoding HRM can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide or fragment thereof which encodes HRM. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector and even longer if replication elements are part of the vector system. [0221]
  • As mentioned above, modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5′ or regulatory regions of the gene encoding HRM (signal sequence, promoters, enhancers, and introns). Oligonucleotides derived from the transcription initiation site, e.g., between positions −10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using “triple helix” base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee et al. (1994) In: Huber and Carr, [0222] Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco N.Y.). The complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples which may be used include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding HRM. [0223]
  • Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. [0224]
  • Complementary ribonucleic acid molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding HRM. Such DNA sequences may be incorporated into a wide variety of vectors with 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. [0225]
  • 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. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases. [0226]
  • Many methods for introducing vectors into cells or tissues are available for use in vivo, in vitro, and ex vivo. For ex vivo therapy, 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 polycationic amino polymers (Goldman et al. (1997) Nature Biotechnol 15:462-66, incorporated herein by reference) may be achieved using methods which are well known in the art. [0227]
  • Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans. [0228]
  • An additional embodiment of the invention relates to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of HRM, antibodies to HRM, mimetics, agonists, antagonists, or inhibitors of HRM. The compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones. [0229]
  • The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means. [0230]
  • In addition to the active ingredients, these pharmaceutical compositions may contain pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of [0231] Remington's Pharmaceutical Sciences (Mack Publishing, Easton PA).
  • Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient. [0232]
  • Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding auxiliaries, if desired, to obtain tablets or dragee cores. Excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol, starch from corn, wheat, rice, potato, or other plants, cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose, gums including arabic and tragacanth, and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. [0233]
  • Dragee cores may be used in conjunction with coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage. [0234]
  • Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers. [0235]
  • Pharmaceutical formulations for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution. Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as oily injection suspensions. Lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. [0236]
  • For topical or nasal administration, penetrants to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. [0237]
  • The pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. [0238]
  • The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is com buffer prior to use. [0239]
  • After pharmaceutical compositions have been prepared, they can be placed in a container and labeled for treatment of an indicated condition. For administration of HRM, such labeling would include amount, frequency, and method of administration. [0240]
  • Pharmaceutical compositions for use in the invention include compositions wherein the active ingredients are contained in 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. [0241]
  • For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. [0242]
  • A therapeutically effective dose refers to that amount of active ingredient, for example HRM or portions thereof, antibodies of HRM, agonists, antagonists or inhibitors of HRM, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. [0243]
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration. [0244]
  • The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide levels of the active moiety that produce or maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation. [0245]
  • Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, 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 proteins will be specific to particular cells, conditions, locations, etc. [0246]
  • Diagnostic [0247]
  • In another embodiment, antibodies which specifically bind HRM may be used for the diagnosis of conditions or diseases characterized by expression of HRM, or in assays to monitor patients being treated with HRM, agonists, antagonists or inhibitors. The antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for HRM include methods which utilize the antibody and a label to detect HRM in human body fluids or extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by joining them, either covalently or non-covalently, with a reporter molecule. A wide variety of reporter molecules which are known in the art may be used, several of which are described above. [0248]
  • A variety of protocols including ELISA, RIA, and FACS for measuring HRM are known in the art and provide a basis for diagnosing altered or abnormal levels of HRM expression. Normal or standard values for HRM expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to HRM under conditions for complex formation. The amount of standard complex formation may be quantified by various methods, but preferably by photometric means. Quantities of HRM expressed in subject samples are compared with the standard values from control and diseased samples. Deviation between standard and subject values establishes the parameters for diagnosing disease. [0249]
  • In another embodiment of the invention, the polynucleotides encoding HRM may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of HRM may be correlated with disease. The diagnostic assay may be used to distinguish between absence, presence, and excess expression of HRM, and to monitor regulation of HRM levels during therapeutic intervention. [0250]
  • In one aspect, hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding HRM or closely related molecules, may be used to identify nucleic acid sequences which encode HRM. The specificity of the probe, whether it is made from a highly specific region, e.g., 10 unique nucleotides in the 5′ regulatory region, or a less specific region, e.g., especially in the 3′ coding region, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low) will determine whether the probe identifies only naturally occurring sequences encoding HRM, alleles, or related sequences. [0251]
  • Probes may also be used for the detection of related sequences, and should preferably contain at least 50% of the nucleotides from any of the HRM encoding sequences. The hybridization probes of the invention may be DNA or RNA and derived from the polynucleotide of SEQ ID NOs:50-98 or from genomic sequence including promoter, enhancer elements, and introns of the naturally occurring HRM. [0252]
  • Means for producing specific hybridization probes for polynucleotides encoding HRM include the cloning of nucleic acid sequences encoding HRM or HRM derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the RNA polymerases and the labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, radionuclides such as 32P or 35S, or enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like. [0253]
  • Polynucleotides encoding HRM may be used for the diagnosis of conditions, disorders, or diseases which are associated with either increased or decreased expression of HRM. Examples of such conditions or diseases include adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and cancers of the adrenal gland, bladder, bone, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, bone marrow, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus, and immune disorders such as AIDS, Addison's disease, adult respiratory distress syndrome, allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitus, Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, atrophic gastritis, glomerulonephritis, gout, Graves' disease, hypereosinophilia, irritable bowel syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, and thyroiditis. The polynucleotides encoding HRM may be used in Southern or northern analysis, dot blot, or other membrane-based technologies, in PCR technologies, or in dipstick, pin, or other multiformat assays including microarrays to analyze fluids or tissues from patient biopsies to detect altered HRM expression. Such qualitative or quantitative methods are well known in the art. [0254]
  • In a particular aspect, the polynucleotides encoding HRM may be useful in assays that detect activation or induction of various cancers, particularly those mentioned above. The polynucleotides encoding HRM may be labeled by standard methods, and added to a fluid or tissue sample from a patient under conditions for the formation of hybridization complexes. After an incubation period, the sample is washed, the signal is quantitated and compared with a standard value. If the amount of signal in the biopsied or extracted sample is significantly different from that of a comparable control sample, the polynucleotides have hybridized with nucleic acids in the sample, and the presence of differentially expressed polynucleotides encoding HRM in the sample indicates the presence of the disease. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient. [0255]
  • In order to provide a basis for the diagnosis of disease associated with expression of HRM, 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, which encodes HRM, under conditions for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with those from an experiment where a known amount of an isolated polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for disease. Deviation between standard and subject values is used to establish the presence of disease. [0256]
  • Once disease is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to evaluate whether the level of expression in the patient begins to approximate that which is observed in the normal patient. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months. [0257]
  • With respect to cancer, the presence of a relatively high amount of transcript 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 prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ aggressive treatment earlier thereby preventing further progression of the cancer. [0258]
  • Additional diagnostic uses for oligonucleotides designed from the sequences encoding HRM may involve the use of PCR. Such oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably consist of two polynucleotides, one with sense orientation (5′−>3′) and another with antisense (3′<−5′), employed under optimized conditions for identification of a specific gene or condition. The same two oligomers, nested sets of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantitation of closely related DNA or RNA sequences. [0259]
  • Methods which may also be used to quantitate the expression of HRM include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated (Melby et al. (1993) J Immunol Methods, 159:235-244, Duplaa et al. (1993) Anal Biochem 229-236). The speed of quantitation of multiple samples may be accelerated by running the assay in an multiwell format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation. [0260]
  • In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotides may be used as targets on a microarray. The microarray can be used to monitor the expression level of large numbers of genes simultaneously (to produce a transcript image), and to identify genetic variants, mutations and polymorphisms. This information may be used to determine gene function, understanding the genetic basis of disease, diagnosing disease, and in developing and in monitoring the activities of therapeutic agents. [0261]
  • In one embodiment, the microarray is prepared and used according to the methods described in PCT application WO95/11995, Lockhart et al. (1996, Nature Biotechnol 14:1675-1680) and Schena et al. (1996, Proc Natl Acad Sci 93:10614-10619), all of which are incorporated herein in their entirety by reference. [0262]
  • The microarray is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray, it may be preferable to use oligonucleotides which are only 7-10 nucleotides in length. The microarray may contain oligonucleotides which cover the known 5′, or 3′, sequence, or contain sequential oligonucleotides which cover the full length sequence, or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray may be oligonucleotides that are specific to a gene or genes of interest in which at least a fragment of the sequence is known or that are specific to one or more unidentified cDNAs which are common to a particular cell or tissue type or to a normal, developmental, or disease state. In certain situations it may be to use pairs of oligonucleotides on a microarray. The “pairs” will be identical, except for one nucleotide which is located in the center of the sequence. The second oligonucleotide in the pair (mismatched by one) serves as a control. The number of oligonucleotide pairs may range from 2 to one million. [0263]
  • In order to produce oligonucleotides to a known sequence for a microarray, the gene of interest is examined using a computer algorithm which starts at the 5′ or more preferably at the 3′ end of the polynucleotide. The algorithm identifies oligomers of defined length that are unique to the gene, have a GC content within a range for hybridization, and lack predicted secondary structure that may interfere with hybridization. In one aspect, the oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide, or any other solid support. [0264]
  • In another aspect, the oligonucleotides may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference. In another aspect, a gridded array analogous to a dot or slot blot apparatus may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. In yet another aspect, an array may be produced by hand or using available devices, materials, and machines (including multichannel pipetters or robotic instruments) and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other multiple from 2 to one million which lends itself to the efficient use of commercially available instrumentation. [0265]
  • In order to conduct sample analysis using the microarrays, polynucleotides are extracted from a biological sample. The biological samples may be obtained from any bodily fluid (blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. To produce probes, the polynucleotides extracted from the sample are used to produce nucleic acid sequences which are complementary to the nucleic acids on the microarray. If the microarray consists of cDNAs, antisense RNAs (aRNA) are probes. Therefore, in one aspect, mRNA is used to produce cDNA which, in turn and in the presence of fluorescent nucleotides, is used to produce fragment or oligonucleotide aRNA probes. These fluorescently labeled probes are incubated with the microarray so that the probe sequences hybridize to the cDNA oligonucleotides of the microarray. In another aspect, complementary nucleic acid sequences are used as probes and can also include polynucleotides, fragments, complementary, or antisense sequences produced using restriction enzymes, PCR technologies, and oligolabeling kits which are well known in the art. [0266]
  • Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large scale correlation studies or functional analysis of the sequences, mutations, variants, or polymorphisms among samples (Heller et al. (1997) Proc Natl Acad Sci 94:2150-55). [0267]
  • In another embodiment of the invention, the nucleic acid sequences which encode HRM may also be used to generate hybridization probes which are useful for mapping the naturally occurring genomic sequence. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome or to artificial chromosome constructions, such as human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries as reviewed in Price (1993, Blood Rev 7:127-134) and Trask (1991, Trends Genet 7:149-154). [0268]
  • Fluorescent in situ hybridization (FISH as described in Verma et al. (1988) [0269] Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York N.Y.) may be correlated with other physical chromosome mapping techniques and genetic map data. Examples of genetic map data can be found in various scientific journals or at Online Mendelian Inheritance in Man (OMIM). Correlation between the location of the gene encoding HRM on a physical chromosomal map and a specific disease, or predisposition to a specific disease, may help delimit the region of DNA associated with that genetic disease. The polynucleotides of the invention may be used to detect differences in gene sequences between normal, carrier, or affected individuals.
  • In situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms, or parts thereof, by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, for example, AT to 11q22-23 (Gatti et al. (1988) Nature 336:577-580), any sequences mapping to that area may represent associated or regulatory genes for further investigation. The polynucleotide of the invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals. [0270]
  • In another embodiment of the invention, HRM, its catalytic or immunogenic portions or oligopeptides thereof, can be used for screening libraries of compounds in any of a variety of drug screening techniques. The portion employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes, between HRM and the agent being tested, may be measured. [0271]
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having binding affinity to the protein of interest as described in published PCT application WO84/03564. In this method, large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with HRM, or portions thereof, and washed. Bound HRM is then detected by methods well known in the art. Purified HRM can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support. [0272]
  • In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding HRM specifically compete with a test compound for binding HRM. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with HRM. [0273]
  • In additional embodiments, the polynucleotides which encode HRM may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of polynucleotides that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions. [0274]
  • The examples below are provided to illustrate the invention and are not included for the purpose of limiting the invention.[0275]
    • EXAMPLES
  • For purposes of example, the preparation and sequencing of the LNODNOT03 cDNA library, from which Incyte Clones 1572888, 1573677, 1574624, and 1577239 were isolated, is described. Preparation and sequencing of cDNAs in libraries in the LIFESEQ database (Incyte Genomics, Palo Alto CA) have varied over time, and the gradual changes involved use of kits, plasmids, and machinery available at the particular time the library was made and analyzed. [0276]
    • I LNODNOT03 cDNA Library Construction
  • The LNODNOT03 cDNA library was constructed using 1 μg of polyA RNA isolated from lymph node tissue removed from a 67-year-old Caucasian male during a segmental lung resection and bronchoscopy. Microscopic examination showed that the tissue was extensively necrotic with 10% viable tumor. The invasive grade 3/4 squamous cell carcinoma had formed a mass in the right lower lobe of the lung which had invaded into, but not through, the visceral pleura. Focally, the tumor had obliterated the bronchial lumen although the bronchial margin was negative for dysplasia/neoplasm. One of two intrapulmonary, one of four inferior mediastinal (subcarinal), and two of eight superior mediastinal lymph nodes were metastatically involved. Patient history included hemangioma and tobacco use, the patient was taking Doxycycline, a tetracycline, to treat an infection. [0277]
  • The frozen tissue was homogenized and lysed in guanidinium isothiocyanate solution using a POLYTRON homogenizer (Brinkmann Instruments, Westbury N.Y.). The lysate was centrifuged over a 5.7 M CsCl cushion using an SW28 rotor in a L8-70M ultracentrifuge (Beckman Coulter, Fullerton Calif.) for 18 hours at 25,000 rpm at ambient temperature. The RNA was extracted with acid phenol, pH 4.7, precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended in RNAse-free water, and treated with DNAse at 37C. Extraction and precipitation were repeated as before. The MRNA was isolated using the OLIGOTEX kit (Qiagen, Chatsworth Calif.) and used to construct the cDNA library. [0278]
  • The mRNA was handled according to the recommended protocols in the SUPERSCRIPT plasmid system (Life Technologies). The cDNAs were fractionated on a SEPHAROSE CL4B column (APB), and those cDNAs exceeding 400 bp were ligated into pINCY plasmid (Incyte Genomics). The plasmid was subsequently transformed into DH5α competent cells (Life Technologies). [0279]
    • II Isolation and Sequencing of cDNA Clones
  • Plasmid DNA was released from the cells and purified using the REAL Prep 96 plasmid kit (Qiagen). This kit enabled the simultaneous purification of 96 samples in a 96-well block using multi-channel reagent dispensers. The recommended protocol was employed except for the following changes: 1) the bacteria were cultured in 1 ml of sterile TERRIFIC BROTH (BD Biosciences, Sparks MD) with carbenicillin at 25 mg/L and glycerol at 0.4%, 2) after incubation for 19 hours, the cells were lysed with 0.3 ml of lysis buffer and precipitated using isopropanol, and 3) the plasmid pellet was resuspended in 0.1 ml of distilled water. After the last step in the protocol, samples were transferred to a 96-well block for storage at 4 C. [0280]
  • The cDNAs were prepared using a MICROLAB system (Hamilton) in combination with DNA ENGINE thermal cyclers (MJ Research), sequenced by the method of Sanger and Coulson (1975, J Mol Biol 94:441f) using 377 PRISM DNA sequencing systems (ABI), and reading frame was determined. [0281]
    • III Homology Searching of cDNA Clones and Their Deduced Proteins
  • The polynucleotides and/or amino acid sequences of the Sequence Listing were used to query sequences in the GenBank, SwissProt, BLOCKS, and Pima II databases. These databases, which contain previously identified and annotated sequences, were searched for regions of homology using BLAST, which stands for Basic Local Alignment Search Tool (Altschul (1993) J Mol Evol 36:290-300, Altschul et al. (1990) J Mol Biol 215:403-410). [0282]
  • BLAST produced alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST was especially useful in determining exact matches or in identifying homologs which may be of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant) origin. Other algorithms such as the one described in Smith et al. (1992, Protein Engineering 5:35-51) could have been used when dealing with primary sequence patterns and secondary structure gap penalties. The sequences disclosed in this application have lengths of at least 49 nucleotides, and no more than 12% uncalled bases (where N is recorded rather than A, C, G, or T). [0283]
  • The BLAST approach searched for matches between a query sequence and a database sequence. BLAST evaluated the statistical significance of any matches found and reported only those matches that satisfy the user-selected threshold of significance. In this application, threshold was set at 10[0284] −25 for nucleotides and 10−14 for peptides.
  • Incyte polynucleotides were searched against the GenBank databases for primate (pri), rodent (rod), and other mammalian sequences (mam), and deduced amino acid sequences from the same clones were then searched against GenBank functional protein databases, mammalian (mamp), vertebrate (vrtp), and eukaryote (eukp) for homology. [0285]
    • IV Northern Analysis
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled polynucleotide to a membrane on which RNAs from a particular cell type or tissue have been bound (Sambrook, supra). [0286]
  • Analogous computer techniques use BLAST to search for identical or related molecules in nucleotide databases such as GenBank or the LIFESEQ database (Incyte Genomics). This analysis is much faster than multiple, membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or homologous. [0287]
  • The basis of the search is the product score which is defined as:[0288]
  • % sequence identitv ×% maximum BLAST score/100
  • The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1-2% error, and at 70, the match will be exact. Homologous molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules. [0289]
  • The results of northern analysis are reported as a list of libraries in which the transcript encoding HRM occurs. Abundance and percent abundance are also reported. Abundance directly reflects the number of times a particular transcript is represented in a cDNA library, and percent abundance is abundance divided by the total number of sequences examined in the cDNA library. [0290]
    • V Extension of HRM Encoding Polynucleotides
  • The nucleic acid sequence of an Incyte Clone disclosed in the Sequence Listing was used to design oligonucleotide primers for extending a partial sequence to full length. One primer was synthesized to initiate extension in the antisense direction, and the other was synthesized to extend sequence in the sense direction. Primers were used to facilitate the extension of the known sequence “outward” generating amplicons containing new, unknown nucleotide sequence for the region of interest. The initial primers were designed from the cDNA using OLIGO software (Molecular Insights), or another program, to be about 22 to about 30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures of about 68 to about 72 C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided. [0291]
  • Selected human cDNA libraries (Life Technologies) were used to extend the sequence. If more than one extension is necessary or desired, additional sets of primers are designed to further extend the known region. [0292]
  • High fidelity amplification was obtained by following the instructions for the XL-PCR kit (ABI) and thoroughly mixing the enzyme and reaction mix. Beginning with 40 pmol of each primer and the recommended concentrations of all other components of the kit, PCR was performed using the DNA ENGINE thermal cycler (MJ Research) and the following parameters: Step 1, 94C for 1 min (initial denaturation); Step 2, 65C for 1 min; Step 3, 68C for 6 min; Step 4, 94C for 15 sec; Step 5, 65C for 1 min; Step 6, 68C for 7 min; Step 7, repeat step 4-6 for 15 additional cycles; Step 8, 94C for 15 sec 1 min; Step 10, 68C for 7:15 min; Step 11, repeat step 8-10 for 12 cycles; Step 12, 72C for 13, hold at 4 C. [0293]
  • A 5-10 μl aliquot of the reaction mixture was analyzed by electrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gel to determine which reactions were successful in extending the sequence. Bands thought to contain the largest products were excised from the gel, purified using QIAQUICK kit (Qiagen), and trimmed of overhangs using Klenow enzyme to facilitate religation and cloning. [0294]
  • After ethanol precipitation, the products were redissolved in 13 μl of ligation buffer, 1μ iul T4-DNA ligase (15 units) and 1μl T4 polynucleotide kinase were added, and the mixture was incubated at room temperature for 2-3 hours or overnight at 16 C. Competent [0295] E. coli cells (in 40 μl of media) were transformed with 3 μl of ligation mixture and cultured in 80 μl of SOC medium (Sambrook, supra). After incubation for one hour at 37 C, the E coli mixture was plated on Luria Bertani (LB)-agar (Sambrook, supra) containing 2x carbenicillin (Carb). The following day, several colonies were randomly picked from each plate and cultured in 150 μl of liquid LB/2= Carb medium placed in an individual well of a commercially-available, sterile 96-well microtiter plate. The following day, 5 ,μl of each overnight culture was transferred into a non-sterile 96-well plate and after dilution 1:10 with water, 5 μl of each sample was transferred into a PCR array.
  • For PCR amplification, 18 ,μl of concentrated PCR reaction mix (3.3x) containing 4 units of rTth DNA polymerase, a vector primer, and one or both of the gene specific primers used for the extension reaction were added to each well. Amplification was performed using the following conditions: Step 1, 94 C for 60 sec; Step 2, 94 C for 20 sec; Step 3, 55 C for 30 sec; Step 4, 72 C for 90 sec; Step 5, repeat steps 2-4 for an additional 29 cycles, Step 6, 72 C for 180 sec, and Step 7, hold at 4 C. [0296]
  • Aliquots of the PCR reactions were run on agarose gels together with molecular weight markers. The sizes of the PCR products were compared to the original partial cDNAs, and clones were selected, ligated into plasmid, and sequenced. [0297]
  • In like manner, a genomic library and a polynucleotide selected from SEQ ID NOs:50-98 is used to obtain 5′ regulatory sequences using the procedure above. [0298]
    • VI Labeling and Use of Individual Hybridization Probes
  • Hybridization probes derived from SEQ ID NOs:50-98 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base-pairs, is specifically described, the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO software (Molecular Insights), labeled by combining 50 pmol of each oligomer and 250 μCi of [γ-[0299] 32P] adenosine triphosphate (APB) and T4 polynucleotide kinase (NEN Life Science Products, Acton Mass.). The labeled oligonucleotides are purified using SEPHADEX G-25 superfine resin column (APB). A aliquot containing 107 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II, NEN Life Science Products).
  • The DNA from each digest is fractionated on a 0.7 percent agarose gel and transferred to NYTRANPLUS membranes (Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40 C. To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1 × saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film (Eastman Kodak, Rochester N.Y.) is exposed to the blots in a PHOSPHOIMAGER cassette (APB) for several hours, hybridization patterns are compared visually. [0300]
    • VII Microarrays
  • To produce oligonucleotides for a microarray, SEQ ID NOs:50-98 are examined using a computer algorithm which starts at the 3′ end of the polynucleotide. The algorithm identified oligomers of defined length that are unique to the gene, have a GC content within a range for hybridization, and lack predicted secondary structure that would interfere with hybridization. The algorithm identifies approximately 20 sequence-specific oligonucleotides of 20 nucleotides in length (20-mers). A matched set of oligonucleotides are created in which one nucleotide in the center of each sequence is altered. This process is repeated for each gene in the microarray, and double sets of twenty 20 mers are synthesized and arranged on the surface of the silicon chip using a light-directed chemical process (described in PCT/WO95/11995). [0301]
  • In the alternative, a chemical coupling procedure and an ink jet device are used to synthesize oligomers on the surface of a substrate (PCT/WO95/251116). In another alternative, a gridded array is used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical, or chemical bonding procedures. A typical array may be produced by hand or using available materials and machines and contain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots. After hybridization, the microarray is washed to remove nonhybridized probes, and a scanner is used to determine the levels and patterns of fluorescence. The scanned image is examined to determine degree of complementarity and the relative abundance/expression level of each sequence in the microarray. [0302]
    • VIII Complementary Polynucleotides
  • Sequence complementary to the sequence encoding HRM, or any part thereof, is used to detect, decrease or inhibit expression of naturally occurring HRM. Although use of oligonucleotides comprising from about 15 to about 30 base-pairs is described, the same procedure is used with smaller or larger sequence fragments. Oligonucleotides are designed using OLIGO software (Molecular Insights) and the coding sequence of SEQ ID NOs:50-98. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5′ sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the transcript encoding HRM. [0303]
    • IX Expression of HRM
  • Expression of HRM is accomplished by subcloning the cDNAs into vectors and transforming the vectors into host cells. In this case, the cloning vector is also used to express HRM in [0304] E. coli. Upstream of the cloning site, this vector contains a promoter for β-galactosidase, followed by sequence containing the amino-terminal Met, and the subsequent seven residues of β-galactosidase. Immediately following these eight residues is a bacteriophage promoter useful for transcription and a linker containing a number of unique restriction sites.
  • Induction of an isolated, transformed bacterial strain with IPTG using standard methods produces a fusion protein which consists of the first eight residues of β-galactosidase, about 5 to 15 residues of linker, and the full length protein. The signal residues direct the secretion of HRM into the bacterial growth media which can be used directly in the following assay for activity. [0305]
    • X Demonstration of HRM Activity
  • HRM can be expressed in a mammalian cell line such as DLD-1 or HCT116 (ATCC) by transforming the cells with a eukaryotic expression vector encoding HRM. Eukaryotic expression vectors are commercially available and the techniques to introduce them into cells are well known to those skilled in the art. The effect of HRM on cell morphology may be visualized by microscopy, the effect on cell growth may be determined by measuring cell doubling-time, and the effect on tumorigenicity may be assessed by the ability of transformed cells to grow in a soft agar growth assay (Groden (1995) Cancer Res 55:1531-1539). [0306]
    • XI Production of HRM Specific Antibodies
  • HRM that is purified using PAGE electrophoresis (Sambrook, supra), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols. In the alternative, an amino acid sequence deduced from SEQ ID NOs:50-98 is analyzed using LASERGENE software (DNASTAR, Madison Wis.) to determine regions of high immunogenicity, and an oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Selection of epitope, such as those near the C-terminus or in hydrophilic regions, is described by Ausubel (supra). [0307]
  • Typically, the oligopeptides are 15 residues in length, synthesized using a 43 1A Peptide synthesizer (ABI) using Fmoc-chemistry, and coupled to keyhole limpet hemocyanin (Sigma-Aldrich, St. Louis Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (Ausubel supra). Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. The resulting antisera are tested for antipeptide activity, for example, by binding the protein to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio iodinated, goat anti-rabbit IgG. [0308]
    • XII Purification of Naturally Occurring HRM Using Specific Antibodies
  • Naturally occurring or recombinant HRM is substantially purified by immunoaffinity chromatography using antibodies specific for HRM. An immunoaffinity column is constructed by covalently coupling HRM antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE resin (APB). After the coupling, the resin is blocked and washed according to the manufacturer's instructions. [0309]
  • Media containing HRM is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of HRM (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/protein binding (eg, a buffer of pH 2-3 or a high concentration of a chaotrope, such as urea or thiocyanate ion), and HRM is collected. [0310]
    • XIII Identification of Molecules Which Interact with HRM
  • HRM, or biologically active portions thereof, are labeled with [0311] 125I Bolton-Hunter reagent (Bolton et al. (1973) Biochem J 133:529-39). Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled HRM, washed and any wells with labeled HRM complex are assayed. Data obtained using different concentrations of HRM are used to calculate values for the number, affinity, and association of HRM with the candidate molecules.
  • All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims. [0312]
  • 1 98 151 amino acids amino acid single linear U937NOT01 133 1 Met Thr Asn Glu Glu Pro Leu Pro Lys Lys Val Arg Leu Ser Glu 5 10 15 Thr Asp Phe Lys Val Met Ala Arg Asp Glu Leu Ile Leu Arg Trp 20 25 30 Lys Gln Tyr Glu Ala Tyr Val Gln Ala Leu Glu Gly Lys Tyr Thr 35 40 45 Asp Leu Asn Ser Asn Asp Val Thr Gly Leu Arg Glu Ser Glu Glu 50 55 60 Lys Leu Lys Gln Gln Gln Gln Glu Ser Ala Arg Arg Glu Asn Ile 65 70 75 Leu Val Met Arg Leu Ala Thr Lys Glu Gln Glu Met Gln Glu Cys 80 85 90 Thr Thr Gln Ile Gln Tyr Leu Lys Gln Val Gln Gln Pro Ser Val 95 100 105 Ala Gln Leu Arg Ser Thr Met Val Asp Pro Ala Ile Asn Leu Phe 110 115 120 Phe Leu Lys Met Lys Gly Glu Leu Glu Gln Thr Lys Asp Lys Leu 125 130 135 Glu Gln Ala Gln Asn Glu Leu Ser Ala Trp Lys Phe Thr Pro Asp 140 145 150 Arg 185 amino acids amino acid single linear U937NOT01 1762 2 Met Leu Thr Leu Ala Ser Lys Leu Lys Arg Asp Asp Gly Leu Lys 5 10 15 Gly Ser Arg Thr Ala Ala Thr Ala Ser Asp Ser Thr Arg Arg Val 20 25 30 Ser Val Arg Asp Lys Leu Leu Val Lys Glu Val Ala Glu Leu Glu 35 40 45 Ala Asn Leu Pro Cys Thr Cys Lys Val His Phe Pro Asp Pro Asn 50 55 60 Lys Leu His Cys Phe Gln Leu Thr Val Thr Pro Asp Glu Gly Tyr 65 70 75 Tyr Gln Gly Gly Lys Phe Gln Phe Glu Thr Glu Val Pro Asp Ala 80 85 90 Tyr Asn Met Val Pro Pro Lys Val Lys Cys Leu Thr Lys Ile Trp 95 100 105 His Pro Asn Ile Thr Glu Thr Gly Glu Ile Cys Leu Ser Leu Leu 110 115 120 Arg Glu His Ser Ile Asp Gly Thr Gly Trp Ala Pro Thr Arg Thr 125 130 135 Leu Lys Asp Val Val Trp Gly Leu Asn Ser Leu Phe Thr Asp Leu 140 145 150 Leu Asn Phe Asp Asp Pro Leu Asn Ile Glu Ala Ala Glu His His 155 160 165 Leu Arg Asp Lys Glu Asp Phe Arg Asn Lys Val Asp Asp Tyr Ile 170 175 180 Lys Arg Tyr Ala Arg 185 59 amino acids amino acid single linear U937NOT01 1847 3 Met Gly Lys Val Asn Val Ala Lys Leu Arg Tyr Met Ser Arg Asp 5 10 15 Asp Phe Arg Val Leu Thr Ala Val Glu Met Gly Met Lys Asn His 20 25 30 Glu Ile Val Pro Gly Ser Leu Ile Ala Ser Ile Ala Ser Leu Lys 35 40 45 His Gly Gly Cys Asn Lys Val Leu Arg Glu Leu Val Lys His 50 55 338 amino acids amino acid single linear HMC1NOT01 9337 4 Met Leu Glu Thr Phe Gly His Leu Val Ser Val Gly Trp Glu Thr 5 10 15 Thr Leu Glu Asn Lys Glu Leu Ala Pro Asn Ser Asp Ile Pro Glu 20 25 30 Glu Glu Pro Ala Pro Ser Leu Lys Val Gln Glu Ser Ser Arg Asp 35 40 45 Cys Ala Leu Ser Ser Thr Leu Glu Asp Thr Leu Gln Gly Gly Val 50 55 60 Gln Glu Val Gln Asp Thr Val Leu Lys Gln Met Glu Ser Ala Gln 65 70 75 Glu Lys Asp Leu Pro Gln Lys Lys His Phe Asp Asn Arg Glu Ser 80 85 90 Gln Ala Asn Ser Gly Ala Leu Asp Thr Asn Gln Val Ser Leu Gln 95 100 105 Lys Ile Asp Asn Pro Glu Ser Gln Ala Asn Ser Gly Ala Leu Asp 110 115 120 Thr Asn Gln Val Leu Leu His Lys Ile Pro Pro Arg Lys Arg Leu 125 130 135 Arg Lys Arg Asp Ser Gln Val Lys Ser Met Lys His Asn Ser Arg 140 145 150 Val Lys Ile His Gln Lys Ser Cys Glu Arg Gln Lys Ala Lys Glu 155 160 165 Gly Asn Gly Cys Arg Lys Thr Phe Ser Arg Ser Thr Lys Gln Ile 170 175 180 Thr Phe Ile Arg Ile His Lys Gly Ser Gln Val Cys Arg Cys Ser 185 190 195 Glu Cys Gly Lys Ile Phe Arg Asn Pro Arg Tyr Phe Ser Val His 200 205 210 Lys Lys Ile His Thr Gly Glu Arg Pro Tyr Val Cys Gln Asp Cys 215 220 225 Gly Lys Gly Phe Val Gln Ser Ser Ser Leu Thr Gln His Gln Arg 230 235 240 Val His Ser Gly Glu Arg Pro Phe Glu Cys Gln Glu Cys Gly Arg 245 250 255 Thr Phe Asn Asp Arg Ser Ala Ile Ser Gln His Leu Arg Thr His 260 265 270 Thr Gly Ala Lys Pro Tyr Lys Cys Gln Asp Cys Gly Lys Ala Phe 275 280 285 Arg Gln Ser Ser His Leu Ile Arg His Gln Arg Thr His Thr Gly 290 295 300 Glu Arg Pro Tyr Ala Cys Asn Lys Cys Gly Lys Ala Phe Thr Gln 305 310 315 Ser Ser His Leu Ile Gly His Gln Arg Thr His Asn Arg Thr Lys 320 325 330 Arg Lys Lys Lys Gln Pro Thr Ser 335 456 amino acids amino acid single linear HMC1NOT01 9476 5 Met Lys Ile Glu Glu Val Lys Ser Thr Thr Lys Thr Gln Arg Ile 5 10 15 Ala Ser His Ser His Val Lys Gly Leu Gly Leu Asp Glu Ser Gly 20 25 30 Leu Ala Lys Gln Ala Ala Ser Gly Leu Val Gly Gln Glu Asn Ala 35 40 45 Arg Glu Ala Cys Gly Val Ile Val Glu Leu Ile Glu Ser Lys Lys 50 55 60 Met Ala Gly Arg Ala Val Leu Leu Ala Gly Pro Pro Gly Thr Gly 65 70 75 Lys Thr Ala Leu Ala Leu Ala Ile Ala Gln Glu Leu Gly Ser Lys 80 85 90 Val Pro Phe Cys Pro Met Val Gly Ser Glu Val Tyr Ser Thr Glu 95 100 105 Ile Lys Lys Thr Glu Val Leu Met Glu Asn Phe Arg Arg Ala Ile 110 115 120 Gly Leu Arg Ile Lys Glu Thr Lys Glu Val Tyr Glu Gly Glu Val 125 130 135 Thr Glu Leu Thr Pro Cys Glu Thr Glu Asn Pro Met Gly Gly Tyr 140 145 150 Gly Lys Thr Ile Ser His Val Ile Ile Gly Leu Lys Thr Ala Lys 155 160 165 Gly Thr Lys Gln Leu Lys Leu Asp Pro Ser Ile Phe Glu Ser Leu 170 175 180 Gln Lys Glu Arg Val Glu Ala Gly Asp Val Ile Tyr Ile Glu Ala 185 190 195 Asn Ser Gly Ala Val Lys Arg Gln Gly Arg Cys Asp Thr Tyr Ala 200 205 210 Thr Glu Phe Asp Leu Glu Ala Glu Glu Tyr Val Pro Leu Pro Lys 215 220 225 Gly Asp Val His Lys Lys Lys Glu Ile Ile Gln Asp Val Thr Leu 230 235 240 His Asp Leu Asp Val Ala Asn Ala Arg Pro Gln Gly Gly Gln Asp 245 250 255 Ile Leu Ser Met Met Gly Gln Leu Met Lys Pro Lys Lys Thr Glu 260 265 270 Ile Thr Asp Lys Leu Arg Gly Glu Ile Asn Lys Val Val Asn Lys 275 280 285 Tyr Ile Asp Gln Gly Ile Ala Glu Leu Val Pro Gly Val Leu Phe 290 295 300 Val Asp Glu Val His Met Leu Asp Ile Glu Cys Phe Thr Tyr Leu 305 310 315 His Arg Ala Leu Glu Ser Ser Ile Ala Pro Ile Val Ile Phe Ala 320 325 330 Ser Asn Arg Gly Asn Cys Val Ile Arg Gly Thr Glu Asp Ile Thr 335 340 345 Ser Pro His Gly Ile Pro Leu Asp Leu Leu Asp Arg Val Met Ile 350 355 360 Ile Arg Thr Met Leu Tyr Thr Pro Gln Glu Met Lys Gln Ile Ile 365 370 375 Lys Ile Arg Ala Gln Thr Glu Gly Ile Asn Ile Ser Glu Glu Ala 380 385 390 Leu Asn His Leu Gly Glu Ile Gly Thr Lys Thr Thr Leu Arg Tyr 395 400 405 Ser Val Gln Leu Leu Thr Pro Ala Asn Leu Leu Ala Lys Ile Asn 410 415 420 Gly Lys Asp Ser Ile Glu Lys Glu His Val Glu Glu Ile Ser Glu 425 430 435 Leu Phe Tyr Asp Ala Lys Ser Ser Ala Lys Ile Leu Ala Asp Gln 440 445 450 Gln Asp Lys Tyr Met Lys 455 210 amino acids amino acid single linear THP1PLB01 10370 6 Met Val Leu Trp Leu Lys Gly Val Thr Phe Asn Val Thr Thr Val 5 10 15 Asp Thr Lys Arg Arg Thr Glu Thr Val Gln Lys Leu Cys Pro Gly 20 25 30 Gly Gln Leu Pro Phe Leu Leu Tyr Gly Thr Glu Val His Thr Asp 35 40 45 Thr Asn Lys Ile Glu Glu Phe Leu Glu Ala Val Leu Cys Pro Pro 50 55 60 Arg Tyr Pro Lys Leu Ala Ala Leu Asn Pro Glu Ser Asn Thr Ala 65 70 75 Gly Leu Asp Ile Phe Ala Lys Phe Ser Ala Tyr Ile Lys Asn Ser 80 85 90 Asn Pro Ala Leu Asn Asp Asn Leu Glu Lys Gly Leu Leu Lys Ala 95 100 105 Leu Lys Val Leu Asp Asn Tyr Leu Thr Ser Pro Leu Pro Glu Glu 110 115 120 Val Asp Glu Thr Ser Ala Glu Asp Glu Gly Val Ser Gln Arg Lys 125 130 135 Phe Leu Asp Gly Asn Glu Leu Thr Leu Ala Asp Cys Asn Leu Leu 140 145 150 Pro Lys Leu His Ile Val Gln Val Val Cys Lys Lys Tyr Arg Gly 155 160 165 Phe Thr Ile Pro Glu Ala Phe Arg Gly Val His Arg Tyr Leu Ser 170 175 180 Asn Ala Tyr Ala Arg Glu Glu Phe Ala Ser Thr Cys Pro Asp Asp 185 190 195 Glu Glu Ile Glu Leu Ala Tyr Glu Gln Val Ala Lys Ala Leu Lys 200 205 210 255 amino acids amino acid single linear THP1NOB01 30137 7 Met Leu Gly Gln Leu Leu Pro His Thr Ala Arg Gly Leu Gly Ala 5 10 15 Ala Glu Met Pro Gly Gln Gly Pro Gly Ser Asp Trp Thr Glu Arg 20 25 30 Ser Ser Ser Ala Glu Pro Pro Ala Val Ala Gly Thr Glu Gly Gly 35 40 45 Gly Gly Gly Ser Ala Gly Tyr Ser Cys Tyr Gln Asn Ser Lys Gly 50 55 60 Ser Asp Arg Ile Lys Asp Gly Tyr Lys Val Asn Ser His Ile Ala 65 70 75 Lys Leu Gln Glu Leu Trp Lys Thr Pro Gln Asn Gln Thr Ile His 80 85 90 Leu Ser Lys Ser Met Met Glu Ala Ser Phe Phe Lys His Pro Asp 95 100 105 Leu Thr Thr Gly Gln Lys Arg Tyr Leu Cys Ser Ile Ala Lys Ile 110 115 120 Tyr Asn Ala Asn Tyr Leu Lys Met Leu Met Lys Arg Gln Tyr Met 125 130 135 His Val Leu Gln His Ser Ser Gln Lys Pro Gly Val Leu Thr His 140 145 150 His Arg Ser Arg Leu Ser Ser Arg Tyr Ser Gln Lys Gln His Tyr 155 160 165 Pro Cys Thr Thr Trp Arg His Gln Leu Glu Arg Glu Asp Ser Gly 170 175 180 Ser Ser Asp Ile Ala Ala Ala Ser Ala Pro Glu Met Leu Ile Gln 185 190 195 His Ser Leu Trp Arg Pro Val Arg Asn Lys Glu Gly Ile Lys Thr 200 205 210 Gly Tyr Ala Ser Lys Thr Arg Cys Lys Ser Leu Lys Ile Phe Arg 215 220 225 Arg Pro Arg Lys Leu Phe Met Gln Thr Val Ser Ser Asp Asp Ser 230 235 240 Glu Ser His Met Ser Gly Glu Lys Lys Gly Arg Gly Phe Thr Thr 245 250 255 188 amino acids amino acid single linear SYNORAB01 77180 8 Met Ala Leu Ala Met Leu Val Leu Val Val Ser Pro Trp Ser Ala 5 10 15 Ala Arg Gly Val Leu Arg Asn Tyr Trp Glu Arg Leu Leu Arg Lys 20 25 30 Leu Pro Gln Ser Arg Pro Gly Phe Pro Ser Pro Pro Trp Gly Pro 35 40 45 Ala Leu Ala Val Gln Gly Pro Ala Met Phe Thr Glu Pro Ala Asn 50 55 60 Asp Thr Ser Gly Ser Lys Glu Asn Ser Ser Leu Leu Asp Ser Ile 65 70 75 Phe Trp Met Ala Ala Pro Lys Asn Arg Arg Thr Ile Glu Val Asn 80 85 90 Arg Cys Arg Arg Arg Asn Pro Gln Lys Leu Ile Lys Val Lys Asn 95 100 105 Asn Ile Asp Val Cys Pro Glu Cys Gly His Leu Lys Gln Lys His 110 115 120 Val Leu Cys Ala Tyr Cys Tyr Glu Lys Val Cys Lys Glu Thr Ala 125 130 135 Glu Ile Arg Arg Gln Ile Gly Lys Gln Glu Gly Gly Pro Phe Lys 140 145 150 Ala Pro Thr Ile Glu Thr Val Val Leu Tyr Thr Gly Glu Thr Pro 155 160 165 Ser Glu Gln Asp Gln Gly Lys Arg Ile Ile Glu Arg Asp Arg Lys 170 175 180 Arg Pro Ser Trp Phe Thr Gln Asn 185 531 amino acids amino acid single linear PITUNOR01 98974 9 Met Ala Pro Thr Ile Gln Thr Gln Ala Gln Arg Glu Asp Gly His 5 10 15 Arg Pro Asn Ser His Arg Thr Leu Pro Glu Arg Ser Gly Val Val 20 25 30 Cys Arg Val Lys Tyr Cys Asn Ser Leu Pro Asp Ile Pro Phe Asp 35 40 45 Pro Lys Phe Ile Thr Tyr Pro Phe Asp Gln Asn Arg Phe Val Gln 50 55 60 Tyr Lys Ala Thr Ser Leu Glu Lys Gln His Lys His Asp Leu Leu 65 70 75 Thr Glu Pro Asp Leu Gly Val Thr Ile Asp Leu Ile Asn Pro Asp 80 85 90 Thr Tyr Arg Ile Asp Pro Asn Val Leu Leu Asp Pro Ala Asp Glu 95 100 105 Lys Leu Leu Glu Glu Glu Ile Gln Ala Pro Thr Ser Ser Lys Arg 110 115 120 Ser Gln Gln His Ala Lys Val Val Pro Trp Met Arg Lys Thr Glu 125 130 135 Tyr Ile Ser Thr Glu Phe Asn Arg Tyr Gly Ile Ser Asn Glu Lys 140 145 150 Pro Glu Val Lys Ile Gly Val Ser Val Lys Gln Gln Phe Thr Glu 155 160 165 Glu Glu Ile Tyr Lys Asp Arg Asp Ser Gln Ile Thr Ala Ile Glu 170 175 180 Lys Thr Phe Glu Asp Ala Gln Lys Ser Ile Ser Gln His Tyr Ser 185 190 195 Lys Pro Arg Val Thr Pro Val Glu Val Met Pro Val Phe Pro Asp 200 205 210 Phe Lys Met Trp Ile Asn Pro Cys Ala Gln Val Ile Phe Asp Ser 215 220 225 Asp Pro Ala Pro Lys Asp Thr Ser Gly Ala Ala Ala Leu Glu Met 230 235 240 Met Ser Gln Ala Met Ile Arg Gly Met Met Asp Glu Glu Gly Asn 245 250 255 Gln Phe Val Ala Tyr Phe Leu Pro Val Glu Glu Thr Leu Lys Lys 260 265 270 Arg Lys Arg Asp Gln Glu Glu Glu Met Asp Tyr Ala Pro Asp Asp 275 280 285 Val Tyr Asp Tyr Lys Ile Ala Arg Glu Tyr Asn Trp Asn Val Lys 290 295 300 Asn Lys Ala Ser Lys Gly Tyr Glu Glu Asn Tyr Phe Phe Ile Phe 305 310 315 Arg Glu Gly Asp Gly Val Tyr Tyr Asn Glu Leu Glu Thr Arg Val 320 325 330 Arg Leu Ser Lys Arg Arg Ala Lys Ala Gly Val Gln Ser Gly Thr 335 340 345 Asn Ala Leu Leu Val Val Lys His Arg Asp Met Asn Glu Lys Glu 350 355 360 Leu Glu Ala Gln Glu Ala Arg Lys Ala Gln Leu Glu Asn His Glu 365 370 375 Pro Glu Glu Glu Glu Glu Glu Glu Met Glu Thr Glu Glu Lys Glu 380 385 390 Ala Gly Gly Ser Asp Glu Glu Gln Glu Lys Gly Ser Ser Ser Glu 395 400 405 Lys Glu Gly Ser Glu Asp Glu His Ser Gly Ser Glu Ser Glu Arg 410 415 420 Glu Glu Gly Asp Arg Asp Glu Ala Ser Asp Lys Ser Gly Ser Gly 425 430 435 Glu Asp Glu Ser Ser Glu Asp Glu Ala Arg Ala Ala Arg Asp Lys 440 445 450 Glu Glu Ile Phe Gly Ser Asp Ala Asp Ser Glu Asp Asp Ala Asp 455 460 465 Ser Asp Asp Glu Asp Arg Gly Gln Ala Gln Gly Gly Ser Asp Asn 470 475 480 Asp Ser Asp Ser Gly Ser Asn Gly Gly Gly Gln Arg Ser Arg Ser 485 490 495 His Ser Arg Ser Ala Ser Pro Phe Pro Ser Gly Ser Glu His Ser 500 505 510 Ala Gln Glu Asp Gly Ser Glu Ala Ala Ala Ser Asp Ser Ser Glu 515 520 525 Ala Asp Ser Asp Ser Asp 530 348 amino acids amino acid single linear MUSCNOT01 118160 10 Met Gly Gln Glu Glu Glu Leu Leu Arg Ile Ala Lys Lys Leu Glu 5 10 15 Lys Met Val Ala Arg Lys Asn Thr Glu Gly Ala Leu Asp Leu Leu 20 25 30 Lys Lys Leu His Ser Cys Gln Met Ser Ile Gln Leu Leu Gln Thr 35 40 45 Thr Arg Ile Gly Val Ala Val Asn Gly Val Arg Lys His Cys Ser 50 55 60 Asp Lys Glu Val Val Ser Leu Ala Lys Val Leu Ile Lys Asn Trp 65 70 75 Lys Arg Leu Leu Asp Ser Pro Gly Pro Pro Lys Gly Glu Lys Gly 80 85 90 Glu Glu Arg Glu Lys Ala Lys Lys Lys Glu Lys Gly Leu Glu Cys 95 100 105 Ser Asp Trp Lys Pro Glu Ala Gly Leu Ser Pro Pro Arg Lys Lys 110 115 120 Arg Glu Asp Pro Lys Thr Arg Arg Asp Ser Val Asp Ser Lys Ser 125 130 135 Ser Ala Ser Ser Ser Pro Lys Arg Pro Ser Val Glu Arg Ser Asn 140 145 150 Ser Ser Lys Ser Lys Ala Glu Ser Pro Lys Thr Pro Ser Ser Pro 155 160 165 Leu Thr Pro Thr Phe Ala Ser Ser Met Cys Leu Leu Ala Pro Cys 170 175 180 Tyr Leu Thr Gly Asp Ser Val Arg Asp Lys Cys Val Glu Met Leu 185 190 195 Ser Ala Ala Leu Lys Ala Asp Asp Asp Tyr Lys Asp Tyr Gly Val 200 205 210 Asn Cys Asp Lys Met Ala Ser Glu Ile Glu Asp His Ile Tyr Gln 215 220 225 Glu Leu Lys Ser Thr Asp Met Lys Tyr Arg Asn Arg Val Arg Ser 230 235 240 Arg Ile Ser Asn Leu Lys Asp Pro Arg Asn Pro Gly Leu Arg Arg 245 250 255 Asn Val Leu Ser Gly Ala Ile Ser Ala Gly Leu Ile Ala Lys Met 260 265 270 Thr Ala Glu Glu Met Ala Ser Asp Glu Leu Arg Glu Leu Arg Asn 275 280 285 Ala Met Thr Gln Glu Ala Ile Arg Glu His Gln Met Ala Lys Thr 290 295 300 Gly Gly Thr Thr Thr Asp Leu Phe Gln Cys Ser Lys Cys Lys Lys 305 310 315 Lys Asn Cys Thr Tyr Asn Gln Val Gln Thr Arg Ser Ala Asp Glu 320 325 330 Pro Met Thr Thr Phe Val Leu Cys Asn Glu Cys Gly Asn Arg Trp 335 340 345 Lys Phe Cys 393 amino acids amino acid single linear TLYMNOR01 140516 11 Met Arg Thr Leu Phe Asn Leu Leu Trp Leu Ala Leu Ala Cys Ser 5 10 15 Pro Val His Thr Thr Leu Ser Lys Ser Asp Ala Lys Lys Ala Ala 20 25 30 Ser Lys Thr Leu Leu Glu Lys Ser Gln Phe Ser Asp Lys Pro Val 35 40 45 Gln Asp Arg Gly Leu Val Val Thr Asp Leu Lys Ala Glu Ser Val 50 55 60 Val Leu Glu His Arg Ser Tyr Cys Ser Ala Lys Ala Arg Asp Arg 65 70 75 His Phe Ala Gly Asp Val Leu Gly Tyr Val Thr Pro Trp Asn Ser 80 85 90 His Gly Tyr Asp Val Thr Lys Val Phe Gly Ser Lys Phe Thr Gln 95 100 105 Ile Ser Pro Val Trp Leu Gln Leu Lys Arg Arg Gly Arg Glu Met 110 115 120 Phe Glu Val Thr Gly Leu His Asp Val Asp Gln Gly Trp Met Arg 125 130 135 Ala Val Arg Lys His Ala Lys Gly Leu His Ile Val Pro Arg Leu 140 145 150 Leu Phe Glu Asp Trp Thr Tyr Asp Asp Phe Arg Asn Val Leu Asp 155 160 165 Ser Glu Asp Glu Ile Glu Glu Leu Ser Lys Thr Val Val Gln Val 170 175 180 Ala Lys Asn Gln His Phe Asp Gly Phe Val Val Glu Val Trp Asn 185 190 195 Gln Leu Leu Ser Gln Lys Arg Val Gly Leu Ile His Met Leu Thr 200 205 210 His Leu Ala Glu Ala Leu His Gln Ala Arg Leu Leu Ala Leu Leu 215 220 225 Val Ile Pro Pro Ala Ile Thr Pro Gly Thr Asp Gln Leu Gly Met 230 235 240 Phe Thr His Lys Glu Phe Glu Gln Leu Ala Pro Val Leu Asp Gly 245 250 255 Phe Ser Leu Met Thr Tyr Asp Tyr Ser Thr Ala His Gln Pro Gly 260 265 270 Pro Asn Ala Pro Leu Ser Trp Val Arg Ala Cys Val Gln Val Leu 275 280 285 Asp Pro Lys Ser Lys Trp Arg Ser Lys Ile Leu Leu Gly Leu Asn 290 295 300 Phe Tyr Gly Met Asp Tyr Ala Thr Ser Lys Asp Ala Arg Glu Pro 305 310 315 Val Val Gly Ala Arg Tyr Ile Gln Thr Leu Lys Asp His Arg Pro 320 325 330 Arg Met Val Trp Asp Ser Gln Ala Ser Glu His Phe Phe Glu Tyr 335 340 345 Lys Lys Ser Arg Ser Gly Arg His Val Val Phe Tyr Pro Thr Leu 350 355 360 Lys Ser Leu Gln Val Arg Leu Glu Leu Ala Arg Glu Leu Gly Val 365 370 375 Gly Val Ser Ile Trp Glu Leu Gly Gln Gly Leu Asp Tyr Phe Tyr 380 385 390 Asp Leu Leu 320 amino acids amino acid single linear SPLNNOT02 207452 12 Met Val Gly Tyr Asp Pro Lys Pro Asp Gly Arg Asn Asn Thr Lys 5 10 15 Phe Gln Val Ala Val Ala Gly Ser Val Ser Gly Leu Val Thr Arg 20 25 30 Ala Leu Ile Ser Pro Phe Asp Val Ile Lys Ile Arg Phe Gln Leu 35 40 45 Gln His Glu Arg Leu Ser Arg Ser Asp Pro Ser Ala Lys Tyr His 50 55 60 Gly Ile Leu Gln Ala Ser Arg Gln Ile Leu Gln Glu Glu Gly Pro 65 70 75 Thr Ala Phe Trp Lys Gly His Val Pro Ala Gln Ile Leu Ser Ile 80 85 90 Gly Tyr Gly Ala Val Gln Phe Leu Ser Phe Glu Met Leu Thr Glu 95 100 105 Leu Val His Arg Gly Ser Val Tyr Asp Ala Arg Glu Phe Ser Val 110 115 120 His Phe Val Cys Gly Gly Leu Ala Ala Cys Met Ala Thr Leu Thr 125 130 135 Val His Pro Val Asp Val Leu Arg Thr Arg Phe Ala Ala Gln Gly 140 145 150 Glu Pro Lys Val Tyr Asn Thr Leu Arg His Ala Val Gly Thr Met 155 160 165 Tyr Arg Ser Glu Gly Pro Gln Val Phe Tyr Lys Gly Leu Ala Pro 170 175 180 Thr Leu Ile Ala Ile Phe Pro Tyr Ala Gly Leu Gln Phe Ser Cys 185 190 195 Tyr Ser Ser Leu Lys His Leu Tyr Lys Trp Ala Ile Pro Ala Glu 200 205 210 Gly Lys Lys Asn Glu Asn Leu Gln Asn Leu Leu Cys Gly Ser Gly 215 220 225 Ala Gly Val Ile Ser Lys Thr Leu Thr Tyr Pro Leu Asp Leu Phe 230 235 240 Lys Lys Arg Leu Gln Val Gly Gly Phe Glu His Ala Arg Ala Ala 245 250 255 Phe Gly Gln Val Arg Arg Tyr Lys Gly Leu Met Asp Cys Ala Lys 260 265 270 Gln Val Leu Gln Lys Glu Gly Ala Leu Gly Phe Phe Lys Gly Leu 275 280 285 Ser Pro Ser Leu Leu Lys Ala Ala Leu Ser Thr Gly Phe Met Phe 290 295 300 Phe Ser Tyr Glu Phe Phe Cys Asn Val Phe His Cys Met Asn Arg 305 310 315 Thr Ala Ser Gln Arg 320 343 amino acids amino acid single linear SPLNNOT02 208836 13 Met Ala Glu Gln Leu Ser Pro Gly Lys Ala Val Asp Gln Val Cys 5 10 15 Thr Phe Leu Phe Lys Lys Pro Gly Arg Lys Gly Ala Ala Gly Arg 20 25 30 Arg Lys Arg Pro Ala Cys Asp Pro Glu Pro Gly Glu Ser Gly Ser 35 40 45 Ser Ser Asp Glu Gly Cys Thr Val Val Arg Pro Glu Lys Lys Arg 50 55 60 Val Thr His Asn Pro Met Met Gln Lys Thr Arg Asp Ser Gly Lys 65 70 75 Gln Lys Ala Ala Tyr Gly Asp Leu Ser Ser Glu Glu Glu Glu Glu 80 85 90 Asn Glu Pro Glu Ser Leu Gly Val Val Tyr Lys Ser Thr Arg Ser 95 100 105 Ala Lys Pro Val Gly Pro Glu Asp Met Gly Ala Thr Ala Val Tyr 110 115 120 Glu Leu Asp Thr Glu Lys Glu Arg Asp Ala Gln Ala Ile Phe Glu 125 130 135 Arg Ser Gln Lys Ile Gln Glu Glu Leu Arg Gly Lys Glu Asp Asp 140 145 150 Lys Ile Tyr Arg Gly Ile Asn Asn Tyr Gln Lys Tyr Met Lys Pro 155 160 165 Lys Asp Thr Ser Met Gly Asn Ala Ser Ser Gly Met Val Arg Lys 170 175 180 Gly Pro Ile Arg Ala Pro Glu His Leu Arg Ala Thr Val Arg Trp 185 190 195 Asp Tyr Gln Pro Asp Ile Cys Lys Asp Tyr Lys Glu Thr Gly Phe 200 205 210 Cys Gly Phe Gly Asp Ser Cys Lys Phe Leu His Asp Arg Ser Asp 215 220 225 Tyr Lys His Gly Trp Gln Ile Glu Arg Glu Leu Asp Glu Gly Arg 230 235 240 Tyr Gly Val Tyr Glu Asp Glu Asn Tyr Glu Val Gly Ser Asp Asp 245 250 255 Glu Glu Ile Pro Phe Lys Cys Phe Ile Cys Arg Gln Ser Phe Gln 260 265 270 Asn Pro Val Val Thr Lys Cys Arg His Tyr Phe Cys Glu Ser Cys 275 280 285 Ala Leu Gln His Phe Arg Thr Thr Pro Arg Cys Tyr Val Cys Asp 290 295 300 Gln Gln Thr Asn Gly Val Phe Asn Pro Ala Lys Glu Leu Ile Ala 305 310 315 Lys Leu Glu Lys His Arg Ala Thr Gly Glu Gly Gly Ala Ser Asp 320 325 330 Leu Pro Glu Asp Pro Asp Glu Asp Ala Ile Pro Ile Thr 335 340 368 amino acids amino acid single linear MMLR3DT01 569710 14 Met Ser Ala Gln Ser Val Glu Glu Asp Ser Ile Leu Ile Ile Pro 5 10 15 Thr Pro Asp Glu Glu Glu Lys Ile Leu Arg Val Lys Leu Glu Glu 20 25 30 Asp Pro Asp Gly Glu Glu Gly Ser Ser Ile Pro Trp Asn His Leu 35 40 45 Pro Asp Pro Glu Ile Phe Arg Gln Arg Phe Arg Gln Phe Gly Tyr 50 55 60 Gln Asp Ser Pro Gly Pro Arg Glu Ala Val Ser Gln Leu Arg Glu 65 70 75 Leu Cys Arg Leu Trp Leu Arg Pro Glu Thr His Thr Lys Glu Gln 80 85 90 Ile Leu Glu Leu Val Val Leu Glu Gln Phe Val Ala Ile Leu Pro 95 100 105 Lys Glu Leu Gln Thr Trp Val Arg Asp His His Pro Glu Asn Gly 110 115 120 Glu Glu Ala Val Thr Val Leu Glu Asp Leu Glu Ser Glu Leu Asp 125 130 135 Asp Pro Gly Gln Pro Val Ser Leu Arg Arg Arg Lys Arg Glu Val 140 145 150 Leu Val Glu Asp Met Val Ser Gln Glu Glu Ala Gln Gly Leu Pro 155 160 165 Ser Ser Glu Leu Asp Ala Val Glu Asn Gln Leu Lys Trp Ala Ser 170 175 180 Trp Glu Leu His Ser Leu Arg His Cys Asp Asp Asp Gly Arg Thr 185 190 195 Glu Asn Gly Ala Leu Ala Pro Lys Gln Glu Leu Pro Ser Ala Leu 200 205 210 Glu Ser His Glu Val Pro Gly Thr Leu Ser Met Gly Val Pro Gln 215 220 225 Ile Phe Lys Tyr Gly Glu Thr Cys Phe Pro Lys Gly Arg Phe Glu 230 235 240 Arg Lys Arg Asn Pro Ser Arg Lys Lys Gln His Ile Cys Asp Glu 245 250 255 Cys Gly Lys His Phe Ser Gln Gly Ser Ala Leu Ile Leu His Gln 260 265 270 Arg Ile His Ser Gly Glu Lys Pro Tyr Gly Cys Val Glu Cys Gly 275 280 285 Lys Ala Phe Ser Arg Ser Ser Ile Leu Val Gln His Gln Arg Val 290 295 300 His Thr Gly Glu Lys Pro Tyr Lys Cys Leu Glu Cys Gly Lys Ala 305 310 315 Phe Ser Gln Asn Ser Gly Leu Ile Asn His Gln Arg Ile His Thr 320 325 330 Gly Glu Lys Pro Tyr Glu Cys Val Gln Cys Gly Lys Ser Tyr Ser 335 340 345 Gln Ser Ser Asn Leu Phe Arg His Gln Arg Arg His Asn Ala Glu 350 355 360 Lys Leu Leu Asn Val Val Lys Val 365 158 amino acids amino acid single linear BRSTTUT01 606742 15 Met Glu Gly Pro Arg Arg Gly Pro Glu Val Gly Gly Phe Cys Lys 5 10 15 Tyr Arg Leu Leu Arg Val Ser Arg Ala Leu Cys His Asp Thr Ser 20 25 30 Leu Gly Leu Thr Trp Leu Arg Thr Cys Ser Val Arg Gly Phe Val 35 40 45 Arg Thr Leu Pro Phe Cys Leu Lys Leu Lys Ala Lys Glu Asn Asp 50 55 60 Arg Arg Leu Arg Thr Glu Leu Thr Leu Ala Pro Gly Trp Glu Ala 65 70 75 Ala Ala Leu Leu Asp Ala Thr Tyr Cys Lys Trp Pro Glu Tyr Gln 80 85 90 Arg Gly Gly Phe His Gly Gln Met His Ser Arg Cys Leu Pro Leu 95 100 105 His Leu Asp His Leu Val Val Phe Lys Phe Leu Val Pro Glu Ala 110 115 120 Lys Ser Thr Thr Cys Leu Leu Val Thr Cys Leu Pro Ala Val Val 125 130 135 Val Asp Val Leu Ala Gly Arg Phe Gly Ile Ser His Gln Ser Phe 140 145 150 Cys Thr Val Leu Val Ser Ser Ile 155 334 amino acids amino acid single linear COLNNOT01 611135 16 Met Ala Thr Arg Gln Arg Glu Ser Ser Ile Thr Ser Cys Cys Ser 5 10 15 Thr Ser Ser Cys Asp Ala Asp Asp Glu Gly Val Arg Gly Thr Cys 20 25 30 Glu Asp Ala Ser Leu Cys Lys Arg Phe Ala Val Ser Ile Gly Tyr 35 40 45 Trp His Asp Pro Tyr Ile Gln His Phe Val Arg Leu Ser Lys Glu 50 55 60 Arg Lys Ala Pro Glu Ile Asn Arg Gly Tyr Phe Ala Arg Val His 65 70 75 Gly Val Ser Gln Leu Ile Lys Ala Phe Leu Arg Lys Thr Glu Cys 80 85 90 His Cys Gln Ile Val Asn Leu Gly Ala Gly Met Asp Thr Thr Phe 95 100 105 Trp Arg Leu Lys Asp Glu Asp Leu Leu Pro Ser Lys Tyr Phe Glu 110 115 120 Val Asp Phe Pro Met Ile Val Thr Arg Lys Leu His Ser Ile Lys 125 130 135 Cys Lys Pro Pro Leu Ser Ser Pro Ile Leu Glu Leu His Ser Glu 140 145 150 Asp Thr Leu Gln Met Asp Gly His Ile Leu Asp Ser Lys Arg Tyr 155 160 165 Ala Val Ile Gly Ala Asp Leu Arg Asp Leu Ser Glu Leu Glu Glu 170 175 180 Lys Leu Lys Lys Cys Asn Met Asn Thr Gln Leu Pro Thr Leu Leu 185 190 195 Ile Ala Glu Cys Val Leu Val Tyr Met Thr Pro Glu Gln Ser Ala 200 205 210 Asn Leu Leu Lys Trp Ala Ala Asn Ser Phe Glu Arg Ala Met Phe 215 220 225 Ile Asn Tyr Glu Gln Val Asn Met Gly Asp Arg Phe Gly Gln Ile 230 235 240 Met Ile Glu Asn Leu Arg Arg Arg Gln Cys Asp Leu Ala Gly Val 245 250 255 Glu Thr Cys Lys Ser Leu Glu Ser Gln Lys Glu Arg Leu Leu Ser 260 265 270 Asn Gly Trp Glu Thr Ala Ser Ala Val Asp Met Met Glu Leu Tyr 275 280 285 Asn Arg Leu Pro Arg Ala Glu Val Ser Arg Ile Glu Ser Leu Glu 290 295 300 Phe Leu Asp Glu Met Glu Leu Leu Glu Gln Leu Met Arg His Tyr 305 310 315 Cys Leu Cys Trp Ala Thr Lys Gly Gly Asn Glu Leu Gly Leu Lys 320 325 330 Glu Ile Thr Tyr 488 amino acids amino acid single linear BRSTNOT03 641127 17 Met Ala Ser Thr Ile Thr Gly Ser Gln Asp Cys Ile Val Asn His 5 10 15 Arg Gly Glu Val Asp Gly Glu Pro Glu Leu Asp Ile Ser Pro Cys 20 25 30 Gln Gln Trp Gly Glu Ala Ser Ser Pro Ile Ser Arg Asn Arg Asp 35 40 45 Ser Val Met Thr Leu Gln Ser Gly Cys Phe Glu Asn Ile Glu Ser 50 55 60 Glu Thr Tyr Leu Pro Leu Lys Val Ser Ser Gln Ile Asp Thr Gln 65 70 75 Asp Ser Ser Val Lys Phe Cys Lys Asn Glu Pro Gln Asp His Gln 80 85 90 Glu Ser Arg Arg Leu Phe Val Met Glu Glu Ser Thr Glu Arg Lys 95 100 105 Val Ile Lys Gly Glu Ser Cys Ser Glu Asn Leu Gln Val Lys Leu 110 115 120 Val Ser Asp Gly Gln Glu Leu Ala Ser Pro Leu Leu Asn Gly Glu 125 130 135 Ala Thr Cys Gln Asn Gly Gln Leu Lys Glu Ser Leu Asp Pro Ile 140 145 150 Asp Cys Asn Cys Lys Asp Ile His Gly Trp Lys Ser Gln Val Val 155 160 165 Ser Cys Ser Gln Gln Arg Gly His Thr Glu Glu Lys Pro Cys Asp 170 175 180 His Asn Asn Cys Gly Lys Ile Leu Asn Thr Ser Pro Asp Gly His 185 190 195 Pro Tyr Glu Lys Ile His Thr Ala Glu Lys Gln Tyr Glu Gly Ser 200 205 210 Gln Cys Gly Lys Asn Phe Ser Gln Ser Ser Glu Leu Leu Leu His 215 220 225 Gln Arg Asp His Thr Glu Glu Lys Pro Tyr Lys Cys Glu Gln Cys 230 235 240 Gly Lys Gly Phe Thr Arg Ser Ser Ser Leu Leu Ile His Gln Ala 245 250 255 Val His Thr Asp Glu Lys Pro Tyr Lys Cys Asp Lys Cys Gly Lys 260 265 270 Gly Phe Thr Arg Ser Ser Ser Leu Leu Ile His His Ala Val His 275 280 285 Thr Gly Glu Lys Pro Tyr Lys Cys Asp Lys Cys Gly Lys Gly Phe 290 295 300 Ser Gln Ser Ser Lys Leu His Ile His Gln Arg Val His Thr Gly 305 310 315 Glu Lys Pro Tyr Glu Cys Glu Glu Cys Gly Met Ser Phe Ser Gln 320 325 330 Arg Ser Asn Leu His Ile His Gln Arg Val His Thr Gly Glu Arg 335 340 345 Pro Tyr Lys Cys Gly Glu Cys Gly Lys Gly Phe Ser Gln Ser Ser 350 355 360 Asn Leu His Ile His Arg Cys Ile His Thr Gly Glu Lys Pro Tyr 365 370 375 Gln Cys Tyr Glu Cys Gly Lys Gly Phe Ser Gln Ser Ser Asp Leu 380 385 390 Arg Ile His Leu Arg Val His Thr Gly Glu Lys Pro Tyr His Cys 395 400 405 Gly Lys Cys Gly Lys Gly Phe Ser Gln Ser Ser Lys Leu Leu Ile 410 415 420 His Gln Arg Val His Thr Gly Glu Lys Pro Tyr Glu Cys Ser Lys 425 430 435 Cys Gly Lys Gly Phe Ser Gln Ser Ser Asn Leu His Ile His Gln 440 445 450 Arg Val His Lys Arg Asp Pro Arg Ala His Pro Gly Leu His Ser 455 460 465 Ala His Thr Val Asn Thr Val Lys Tyr Leu Val Ser Leu Leu Leu 470 475 480 Tyr Ile Leu Gln Arg Arg Glu Met 485 255 amino acids amino acid single linear LUNGTUT02 691768 18 Met Gly Arg Asn Lys Lys Lys Lys Arg Asp Gly Asp Asp Arg Arg 5 10 15 Pro Arg Leu Val Leu Ser Phe Asp Glu Glu Lys Arg Arg Glu Tyr 20 25 30 Leu Thr Gly Phe His Lys Arg Lys Val Glu Arg Lys Lys Ala Ala 35 40 45 Ile Glu Glu Ile Lys Gln Arg Leu Lys Glu Glu Gln Arg Lys Leu 50 55 60 Arg Glu Glu Arg His Gln Glu Tyr Leu Lys Met Leu Ala Glu Arg 65 70 75 Glu Glu Ala Leu Glu Glu Ala Asp Glu Leu Asp Arg Leu Val Thr 80 85 90 Ala Lys Thr Glu Ser Val Gln Tyr Asp His Pro Asn His Thr Val 95 100 105 Thr Val Thr Thr Ile Ser Asp Leu Asp Leu Ser Gly Ala Arg Leu 110 115 120 Leu Gly Leu Thr Pro Pro Glu Gly Gly Ala Gly Asp Arg Ser Glu 125 130 135 Glu Glu Ala Ser Ser Thr Glu Lys Pro Thr Lys Ala Leu Pro Arg 140 145 150 Lys Ser Arg Asp Pro Leu Leu Ser Gln Arg Ile Ser Ser Leu Thr 155 160 165 Ala Ser Leu His Ala His Ser Arg Lys Lys Val Lys Arg Lys His 170 175 180 Ser Arg Arg Ala Gln Asp Ser Lys Lys Pro Pro Lys Gly Pro Ser 185 190 195 Tyr Gln Gln Arg Pro Ser Gly Ala Val Phe Thr Gly Lys Ala Pro 200 205 210 Ala Gln Arg Gly Asn Xaa Arg Xaa Glu Asn Glu Ala Gly Cys Pro 215 220 225 His Ser Lys Ala Xaa Arg Gly Xaa Cys Ser Leu Gly Ser Ala Leu 230 235 240 Ala Val Pro Leu Leu Xaa Pro Ala Leu Xaa Leu Lys Val Leu Pro 245 250 255 351 amino acids amino acid single linear SYNOOAT01 724157 19 Met Ala Asp Gln Asp Pro Ala Gly Ile Ser Pro Leu Gln Gln Met 5 10 15 Val Ala Ser Gly Thr Gly Ala Val Val Thr Ser Leu Phe Met Thr 20 25 30 Pro Leu Asp Val Val Lys Val Arg Leu Gln Ser Gln Arg Pro Ser 35 40 45 Met Ala Ser Glu Leu Met Pro Ser Ser Arg Leu Trp Ser Leu Ser 50 55 60 Tyr Thr Lys Trp Lys Cys Leu Leu Tyr Cys Asn Gly Val Leu Glu 65 70 75 Pro Leu Tyr Leu Cys Pro Asn Gly Ala Arg Cys Ala Thr Trp Phe 80 85 90 Gln Asp Pro Thr Arg Phe Thr Gly Thr Met Asp Ala Phe Val Lys 95 100 105 Ile Val Arg His Glu Gly Thr Arg Thr Leu Trp Ser Gly Leu Pro 110 115 120 Ala Thr Leu Val Met Thr Val Pro Ala Thr Ala Ile Tyr Phe Thr 125 130 135 Ala Tyr Asp Gln Leu Lys Ala Phe Leu Cys Gly Arg Ala Leu Thr 140 145 150 Ser Asp Leu Tyr Ala Pro Met Val Ala Gly Ala Leu Ala Arg Leu 155 160 165 Gly Thr Val Thr Val Ile Ser Pro Leu Glu Leu Met Arg Thr Lys 170 175 180 Leu Gln Ala Gln His Val Ser Tyr Arg Glu Leu Gly Ala Cys Val 185 190 195 Arg Thr Ala Val Ala Gln Gly Gly Trp Arg Ser Leu Trp Leu Gly 200 205 210 Trp Gly Pro Thr Ala Leu Arg Asp Val Pro Phe Ser Ala Leu Tyr 215 220 225 Trp Phe Asn Tyr Glu Leu Val Lys Ser Trp Leu Asn Gly Leu Arg 230 235 240 Pro Lys Asp Gln Thr Ser Val Gly Met Ser Phe Val Ala Gly Gly 245 250 255 Ile Ser Gly Thr Val Ala Ala Val Leu Thr Leu Pro Phe Asp Val 260 265 270 Val Lys Thr Gln Arg Gln Val Ala Leu Gly Ala Met Glu Ala Val 275 280 285 Arg Val Asn Pro Leu His Val Asp Ser Thr Trp Leu Leu Leu Arg 290 295 300 Arg Ile Arg Ala Glu Ser Gly Thr Lys Gly Leu Phe Ala Gly Phe 305 310 315 Leu Pro Arg Ile Ile Lys Ala Ala Pro Ser Cys Ala Ile Met Ile 320 325 330 Ser Thr Tyr Glu Phe Gly Lys Ser Phe Phe Gln Arg Leu Asn Gln 335 340 345 Asp Arg Leu Leu Gly Gly 350 535 amino acids amino acid single linear BRAITUT03 864683 20 Met Ser Glu Gly Glu Ser Gln Thr Val Leu Ser Ser Gly Ser Asp 5 10 15 Pro Lys Val Glu Ser Ser Ser Ser Ala Pro Gly Leu Thr Ser Val 20 25 30 Ser Pro Pro Val Thr Ser Thr Thr Ser Ala Ala Ser Pro Glu Glu 35 40 45 Glu Glu Glu Ser Glu Asp Glu Ser Glu Ile Leu Glu Glu Ser Pro 50 55 60 Cys Gly Arg Trp Gln Lys Arg Arg Glu Glu Val Asn Gln Arg Asn 65 70 75 Val Pro Gly Ile Asp Ser Ala Tyr Leu Ala Met Asp Thr Glu Glu 80 85 90 Gly Val Glu Val Val Trp Asn Glu Val Gln Phe Ser Glu Arg Lys 95 100 105 Asn Tyr Lys Leu Gln Glu Glu Lys Val Arg Ala Val Phe Asp Asn 110 115 120 Leu Ile Gln Leu Glu His Leu Asn Ile Val Lys Phe His Lys Tyr 125 130 135 Trp Ala Asp Ile Lys Glu Asn Lys Ala Arg Val Ile Phe Ile Thr 140 145 150 Glu Tyr Met Ser Ser Gly Ser Leu Lys Gln Phe Leu Lys Lys Thr 155 160 165 Lys Lys Asn His Lys Thr Met Asn Glu Lys Ala Trp Lys Arg Trp 170 175 180 Cys Thr Gln Ile Leu Ser Ala Leu Ser Tyr Leu His Ser Cys Asp 185 190 195 Pro Pro Ile Ile His Gly Asn Leu Thr Cys Asp Thr Ile Phe Ile 200 205 210 Gln His Asn Gly Leu Ile Lys Ile Gly Ser Val Ala Pro Asp Thr 215 220 225 Ile Asn Asn His Val Lys Thr Cys Arg Glu Glu Gln Lys Asn Leu 230 235 240 His Phe Phe Ala Pro Glu Tyr Gly Glu Val Thr Asn Val Thr Thr 245 250 255 Ala Val Asp Ile Tyr Ser Phe Gly Met Cys Ala Leu Glu Met Ala 260 265 270 Val Leu Glu Ile Gln Gly Asn Gly Glu Ser Ser Tyr Val Pro Gln 275 280 285 Glu Ala Ile Ser Ser Ala Ile Gln Leu Leu Glu Asp Pro Leu Gln 290 295 300 Arg Glu Phe Ile Gln Lys Cys Leu Gln Ser Glu Pro Ala Arg Arg 305 310 315 Pro Thr Ala Arg Glu Leu Leu Phe His Pro Ala Leu Phe Glu Val 320 325 330 Pro Ser Leu Lys Leu Leu Ala Ala His Cys Ile Val Gly His Gln 335 340 345 His Met Ile Pro Glu Asn Ala Leu Glu Glu Ile Thr Lys Asn Met 350 355 360 Asp Thr Ser Ala Val Leu Ala Glu Ile Pro Ala Gly Pro Gly Arg 365 370 375 Glu Pro Val Gln Thr Leu Tyr Ser Gln Ser Pro Ala Leu Glu Leu 380 385 390 Asp Lys Phe Leu Glu Asp Val Arg Asn Gly Ile Tyr Pro Leu Thr 395 400 405 Ala Phe Gly Leu Pro Arg Pro Gln Gln Pro Gln Gln Glu Glu Val 410 415 420 Thr Ser Pro Val Val Pro Pro Ser Val Lys Thr Pro Thr Pro Glu 425 430 435 Pro Ala Glu Val Glu Thr Arg Lys Val Val Leu Met Gln Cys Asn 440 445 450 Ile Glu Ser Val Glu Glu Gly Val Lys His His Leu Thr Leu Leu 455 460 465 Leu Lys Leu Glu Asp Lys Leu Asn Arg His Leu Ser Cys Asp Leu 470 475 480 Met Pro Asn Glu Asn Ile Pro Glu Leu Ala Ala Glu Leu Val Gln 485 490 495 Leu Gly Phe Ile Ser Glu Ala Asp Gln Ser Arg Leu Thr Ser Leu 500 505 510 Leu Glu Glu Thr Leu Asn Lys Phe Asn Phe Ala Arg Asn Ser Thr 515 520 525 Leu Asn Ser Ala Ala Val Thr Val Ser Ser 530 535 201 amino acids amino acid single linear CERVNOT01 933353 21 Met Ala Ala Thr Ala Leu Leu Glu Ala Gly Leu Ala Arg Val Leu 5 10 15 Phe Tyr Pro Thr Leu Leu Tyr Thr Leu Phe Arg Gly Lys Val Pro 20 25 30 Gly Arg Ala His Arg Asp Trp Tyr His Arg Ile Asp Pro Thr Val 35 40 45 Leu Leu Gly Ala Leu Pro Leu Arg Ser Leu Thr Arg Gln Leu Val 50 55 60 Gln Asp Glu Asn Val Arg Gly Val Ile Thr Met Asn Glu Glu Tyr 65 70 75 Glu Thr Arg Phe Leu Cys Asn Ser Ser Gln Glu Trp Lys Arg Leu 80 85 90 Gly Val Glu Gln Leu Arg Leu Ser Thr Val Asp Met Thr Gly Ile 95 100 105 Pro Thr Leu Asp Asn Leu Gln Lys Gly Val Gln Phe Ala Leu Lys 110 115 120 Tyr Gln Ser Leu Gly Gln Cys Val Tyr Val His Cys Lys Ala Gly 125 130 135 Arg Ser Arg Ser Ala Thr Met Val Ala Ala Tyr Leu Ile Gln Val 140 145 150 His Lys Trp Ser Pro Glu Glu Ala Val Arg Ala Ile Ala Lys Ile 155 160 165 Arg Ser Tyr Ile His Ile Arg Pro Gly Gln Leu Asp Val Leu Lys 170 175 180 Glu Phe His Lys Gln Ile Thr Ala Arg Ala Thr Lys Asp Gly Thr 185 190 195 Phe Val Ile Ser Lys Thr 200 239 amino acids amino acid single linear LATRTUT02 1404643 22 Met Ala Tyr Gln Ser Leu Arg Leu Glu Tyr Leu Gln Ile Pro Pro 5 10 15 Val Ser Arg Ala Tyr Thr Thr Ala Cys Val Leu Thr Thr Ala Ala 20 25 30 Val Gln Leu Glu Leu Ile Thr Pro Phe Gln Leu Tyr Phe Asn Pro 35 40 45 Glu Leu Ile Phe Lys His Phe Gln Ile Trp Arg Leu Ile Thr Asn 50 55 60 Phe Leu Phe Phe Gly Pro Val Gly Phe Asn Phe Leu Phe Asn Met 65 70 75 Ile Phe Leu Tyr Arg Tyr Cys Arg Met Leu Glu Glu Gly Ser Phe 80 85 90 Arg Gly Arg Thr Ala Asp Phe Val Phe Met Phe Leu Phe Gly Gly 95 100 105 Phe Leu Met Thr Leu Phe Gly Leu Phe Val Ser Leu Val Phe Leu 110 115 120 Gly Gln Ala Phe Thr Ile Met Leu Val Tyr Val Trp Ser Arg Arg 125 130 135 Asn Pro Tyr Val Arg Met Asn Phe Phe Gly Leu Leu Asn Phe Gln 140 145 150 Ala Pro Phe Leu Pro Trp Val Leu Met Gly Phe Ser Leu Leu Leu 155 160 165 Gly Asn Ser Ile Ile Val Asp Leu Leu Gly Ile Ala Val Gly His 170 175 180 Ile Tyr Phe Phe Leu Glu Asp Val Phe Pro Asn Gln Pro Gly Gly 185 190 195 Ile Arg Ile Leu Lys Thr Pro Ser Ile Leu Lys Ala Ile Phe Asp 200 205 210 Thr Pro Asp Glu Asp Pro Asn Tyr Asn Pro Leu Pro Glu Glu Arg 215 220 225 Pro Gly Gly Phe Ala Trp Gly Glu Gly Gln Arg Leu Gly Gly 230 235 244 amino acids amino acid single linear SPLNNOT04 1561587 23 Met Met Arg Thr Gln Cys Leu Leu Gly Leu Arg Thr Phe Val Ala 5 10 15 Phe Ala Ala Lys Leu Trp Ser Phe Phe Ile Tyr Leu Leu Arg Arg 20 25 30 Gln Ile Arg Thr Val Ile Gln Tyr Gln Thr Val Arg Tyr Asp Ile 35 40 45 Leu Pro Leu Ser Pro Val Ser Arg Asn Arg Leu Ala Gln Val Lys 50 55 60 Arg Lys Ile Leu Val Leu Asp Leu Asp Glu Thr Leu Ile His Ser 65 70 75 His His Asp Gly Val Leu Arg Pro Thr Val Arg Pro Gly Thr Pro 80 85 90 Pro Asp Phe Ile Leu Lys Val Val Ile Asp Lys His Pro Val Arg 95 100 105 Phe Phe Val His Lys Arg Pro His Val Asp Phe Phe Leu Glu Val 110 115 120 Val Ser Gln Trp Tyr Glu Leu Val Val Phe Thr Ala Ser Met Glu 125 130 135 Ile Tyr Gly Ser Ala Val Ala Asp Lys Leu Asp Asn Ser Arg Ser 140 145 150 Ile Leu Lys Arg Arg Tyr Tyr Arg Gln His Cys Thr Leu Glu Leu 155 160 165 Gly Ser Tyr Ile Lys Asp Leu Ser Val Val His Ser Asp Leu Ser 170 175 180 Ser Ile Val Ile Leu Asp Asn Ser Pro Gly Ala Tyr Arg Ser His 185 190 195 Pro Asp Asn Ala Ile Pro Ile Lys Ser Trp Phe Ser Asp Pro Ser 200 205 210 Asp Thr Ala Leu Leu Asn Leu Leu Pro Met Leu Asp Ala Leu Arg 215 220 225 Phe Thr Ala Asp Val Arg Ser Val Leu Ser Arg Asn Leu His Gln 230 235 240 His Arg Leu Trp 431 amino acids amino acid single linear UTRSNOT05 1568361 24 Met Ser Ser Val Glu Glu Asp Asp Tyr Asp Thr Leu Thr Asp Ile 5 10 15 Asp Ser Asp Lys Asn Val Ile Arg Thr Lys Gln Tyr Leu Tyr Val 20 25 30 Ala Asp Leu Ala Arg Lys Asp Lys Arg Val Leu Arg Lys Lys Tyr 35 40 45 Gln Ile Tyr Phe Trp Asn Ile Ala Thr Ile Ala Val Phe Tyr Ala 50 55 60 Leu Pro Val Val Gln Leu Val Ile Thr Tyr Gln Thr Val Val Asn 65 70 75 Val Thr Gly Asn Gln Asp Ile Cys Tyr Tyr Asn Phe Leu Cys Ala 80 85 90 His Pro Leu Gly Asn Leu Ser Ala Phe Asn Asn Ile Leu Ser Asn 95 100 105 Leu Gly Tyr Ile Leu Leu Gly Leu Leu Phe Leu Leu Ile Ile Leu 110 115 120 Gln Arg Glu Ile Asn His Asn Arg Ala Leu Leu Arg Asn Asp Leu 125 130 135 Cys Ala Leu Glu Cys Gly Ile Pro Lys His Phe Gly Leu Phe Tyr 140 145 150 Ala Met Gly Thr Ala Leu Met Met Glu Gly Leu Leu Ser Ala Cys 155 160 165 Tyr His Val Cys Pro Asn Tyr Thr Asn Phe Gln Phe Asp Thr Ser 170 175 180 Phe Met Tyr Met Ile Ala Gly Leu Cys Met Leu Lys Leu Tyr Gln 185 190 195 Lys Arg His Pro Asp Ile Asn Ala Ser Ala Tyr Ser Ala Tyr Ala 200 205 210 Cys Leu Ala Ile Val Ile Phe Xaa Ser Val Leu Gly Val Val Phe 215 220 225 Gly Lys Gly Asn Thr Ala Phe Trp Ile Val Phe Ser Ile Ile His 230 235 240 Ile Ile Ala Thr Leu Leu Leu Ser Thr Gln Leu Tyr Tyr Met Gly 245 250 255 Arg Trp Lys Leu Asp Ser Gly Ile Phe Arg Arg Ile Leu His Val 260 265 270 Leu Tyr Thr Asp Cys Ile Arg Gln Cys Ser Gly Pro Leu Tyr Val 275 280 285 Asp Arg Met Val Leu Leu Val Met Gly Asn Val Ile Asn Trp Ser 290 295 300 Leu Ala Ala Tyr Gly Leu Ile Met Arg Pro Asn Asp Phe Ala Ser 305 310 315 Tyr Leu Leu Ala Ile Gly Ile Cys Asn Leu Leu Leu Tyr Phe Ala 320 325 330 Phe Tyr Ile Ile Met Lys Leu Arg Ser Gly Glu Arg Ile Lys Leu 335 340 345 Ile Pro Leu Leu Cys Ile Val Cys Thr Ser Val Val Trp Gly Phe 350 355 360 Ala Leu Phe Phe Phe Phe Gln Gly Leu Ser Thr Trp Gln Lys Thr 365 370 375 Pro Ala Glu Ser Arg Glu His Asn Arg Asp Cys Ile Leu Leu Asp 380 385 390 Phe Phe Asp Asp His Asp Ile Trp His Phe Leu Ser Ser Ile Ala 395 400 405 Met Phe Gly Ser Phe Leu Val Leu Leu Thr Leu Asp Asp Asp Leu 410 415 420 Asp Thr Val Gln Arg Asp Lys Ile Tyr Val Phe 425 430 376 amino acids amino acid single linear LNODNOT03 1572888 25 Met Gly His Arg Phe Leu Arg Gly Leu Leu Thr Leu Leu Leu Pro 5 10 15 Pro Pro Pro Leu Tyr Thr Arg His Arg Met Leu Gly Pro Glu Ser 20 25 30 Val Pro Pro Pro Lys Arg Ser Arg Ser Lys Leu Met Ala Pro Pro 35 40 45 Arg Ile Gly Thr His Asn Gly Thr Phe His Cys Asp Glu Ala Leu 50 55 60 Ala Cys Ala Leu Leu Arg Leu Leu Pro Glu Tyr Arg Asp Ala Glu 65 70 75 Ile Val Arg Thr Arg Asp Pro Glu Lys Leu Ala Ser Cys Asp Ile 80 85 90 Val Val Asp Val Gly Gly Glu Tyr Asp Pro Arg Arg His Arg Tyr 95 100 105 Asp His His Gln Arg Ser Phe Thr Glu Thr Met Ser Ser Leu Ser 110 115 120 Pro Gly Lys Pro Trp Gln Thr Lys Leu Ser Ser Ala Gly Leu Ile 125 130 135 Tyr Leu His Phe Gly His Lys Leu Leu Ala Gln Leu Leu Gly Thr 140 145 150 Ser Glu Glu Asp Ser Met Val Gly Thr Leu Tyr Asp Lys Met Tyr 155 160 165 Glu Asn Phe Val Glu Glu Val Asp Ala Val Asp Asn Gly Ile Ser 170 175 180 Gln Trp Ala Glu Gly Glu Pro Arg Tyr Ala Leu Thr Thr Thr Leu 185 190 195 Ser Ala Arg Val Ala Arg Leu Asn Pro Thr Trp Asn His Pro Asp 200 205 210 Gln Asp Thr Glu Ala Gly Phe Lys Arg Ala Met Asp Leu Val Gln 215 220 225 Glu Glu Phe Leu Gln Arg Leu Asp Phe Tyr Gln His Ser Trp Leu 230 235 240 Pro Ala Arg Ala Leu Val Glu Glu Ala Leu Ala Gln Arg Phe Gln 245 250 255 Val Asp Pro Ser Gly Glu Ile Val Glu Leu Ala Lys Gly Ala Cys 260 265 270 Pro Trp Lys Glu His Leu Tyr His Leu Glu Ser Gly Leu Ser Pro 275 280 285 Pro Val Ala Ile Phe Phe Val Ile Tyr Thr Asp Gln Ala Gly Gln 290 295 300 Trp Arg Ile Gln Cys Val Pro Lys Glu Pro His Ser Phe Gln Ser 305 310 315 Arg Leu Pro Leu Pro Glu Pro Trp Arg Gly Leu Arg Asp Glu Ala 320 325 330 Leu Asp Gln Val Ser Gly Ile Pro Gly Cys Ile Phe Val His Ala 335 340 345 Ser Gly Phe Ile Gly Gly His Arg Thr Arg Glu Gly Ala Leu Ser 350 355 360 Met Ala Arg Ala Thr Leu Ala Gln Arg Ser Tyr Leu Pro Gln Ile 365 370 375 Ser 340 amino acids amino acid single linear LNODNOT03 1573677 26 Met Arg Leu Arg Gly Leu Leu Gln Gly Thr Leu Arg Phe His Thr 5 10 15 Ser Pro Pro Thr Asp Ser Ser Val Thr Glu Thr Ile Ile Leu Cys 20 25 30 Thr Met Leu Phe Leu Gly Ser Leu Gly Ala Trp Gly Thr Thr Ser 35 40 45 Ile Ser Thr Gly Ser Ile Phe Ser Leu Lys Thr Leu Arg Ser Gln 50 55 60 His Gly Gly Gln Val Gly Leu Lys Val Ser Arg Pro Arg Ala Gln 65 70 75 Pro Leu Pro Ala Gln Pro Pro Ala Leu Ala Gln Pro Gln Tyr Gln 80 85 90 Ser Pro Gln Gln Pro Pro Gln Thr Arg Trp Val Ala Pro Arg Asn 95 100 105 Arg Asn Ala Ala Phe Gly Gln Ser Gly Gly Ala Gly Ser Asp Ser 110 115 120 Asn Ser Pro Gly Asn Val Gln Pro Asn Ser Ala Pro Ser Val Glu 125 130 135 Ser His Pro Val Leu Glu Lys Leu Lys Ala Ala His Ser Tyr Asn 140 145 150 Pro Lys Glu Phe Glu Trp Asn Leu Lys Ser Gly Arg Val Phe Ile 155 160 165 Ile Lys Ser Tyr Ser Glu Asp Asp Ile His Arg Ser Ile Lys Tyr 170 175 180 Ser Ile Trp Cys Ser Thr Glu His Gly Asn Lys Arg Leu Asp Ser 185 190 195 Ala Phe Arg Cys Met Ser Ser Lys Gly Pro Val Tyr Leu Leu Phe 200 205 210 Ser Val Asn Gly Ser Gly His Phe Cys Gly Val Ala Glu Met Lys 215 220 225 Ser Pro Val Asp Tyr Gly Thr Ser Ala Gly Val Trp Ser Gln Asp 230 235 240 Lys Trp Lys Gly Lys Phe Asp Val Gln Trp Ile Phe Val Lys Asp 245 250 255 Val Pro Asn Asn Gln Leu Arg His Ile Arg Leu Glu Asn Asn Asp 260 265 270 Asn Lys Pro Val Thr Asn Ser Arg Asp Thr Gln Glu Val Pro Leu 275 280 285 Glu Lys Ala Lys Gln Val Leu Lys Ile Ile Ser Ser Tyr Lys His 290 295 300 Thr Thr Ser Ile Phe Asp Asp Phe Ala His Tyr Glu Lys Arg Gln 305 310 315 Arg Arg Arg Arg Trp Cys Ala Arg Asn Gly Arg Val Glu Thr Asn 320 325 330 Asn Glu Gly Glu Pro Val Ser Tyr Met Phe 335 340 174 amino acids amino acid single linear LNODNOT03 1574624 27 Met Ala Asp Val Leu Asp Leu His Glu Ala Gly Gly Glu Asp Phe 5 10 15 Ala Met Asp Glu Asp Gly Asp Glu Ser Ile His Lys Leu Lys Glu 20 25 30 Lys Ala Lys Lys Arg Lys Gly Arg Gly Phe Gly Ser Glu Glu Gly 35 40 45 Ser Arg Ala Arg Met Arg Glu Asp Tyr Asp Ser Val Glu Gln Asp 50 55 60 Gly Asp Glu Pro Gly Pro Gln Arg Ser Val Glu Gly Trp Ile Leu 65 70 75 Phe Val Thr Gly Val His Glu Glu Ala Thr Glu Glu Asp Ile His 80 85 90 Asp Lys Phe Ala Glu Tyr Gly Glu Ile Lys Asn Ile His Leu Asn 95 100 105 Leu Asp Arg Arg Thr Gly Tyr Leu Lys Gly Tyr Thr Leu Val Glu 110 115 120 Tyr Glu Thr Tyr Lys Glu Ala Gln Ala Ala Met Glu Gly Leu Asn 125 130 135 Gly Gln Asp Leu Met Gly Gln Pro Ile Ser Val Asp Trp Cys Phe 140 145 150 Val Arg Gly Pro Pro Lys Gly Lys Arg Arg Gly Gly Arg Arg Arg 155 160 165 Ser Arg Ser Pro Asp Arg Arg Arg Arg 170 179 amino acids amino acid single linear LNODNOT03 1577239 28 Met Val Gln Ala Trp Tyr Met Asp Asp Ala Pro Gly Asp Pro Arg 5 10 15 Gln Pro His Arg Pro Asp Pro Gly Arg Pro Val Gly Leu Glu Gln 20 25 30 Leu Arg Arg Leu Gly Val Leu Tyr Trp Lys Leu Asp Ala Asp Lys 35 40 45 Tyr Glu Asn Asp Pro Glu Leu Glu Lys Ile Arg Arg Glu Arg Asn 50 55 60 Tyr Ser Trp Met Asp Ile Ile Thr Ile Cys Lys Asp Lys Leu Pro 65 70 75 Asn Tyr Glu Glu Lys Ile Lys Met Phe Tyr Glu Glu His Leu His 80 85 90 Leu Asp Asp Glu Ile Arg Tyr Ile Leu Asp Gly Ser Gly Tyr Phe 95 100 105 Asp Val Arg Asp Lys Glu Asp Gln Trp Ile Arg Ile Phe Met Glu 110 115 120 Lys Gly Asp Met Val Thr Leu Pro Ala Gly Ile Tyr His Arg Phe 125 130 135 Thr Val Asp Glu Lys Asn Tyr Thr Lys Ala Met Arg Leu Phe Val 140 145 150 Gly Glu Pro Val Trp Thr Ala Tyr Asn Arg Pro Ala Asp His Phe 155 160 165 Glu Ala Arg Gly Gln Tyr Val Lys Phe Leu Ala Gln Thr Ala 170 175 205 amino acids amino acid single linear BLADNOT03 1598203 29 Met Ala Ala Ala Arg Pro Ser Leu Gly Arg Val Leu Pro Gly Ser 5 10 15 Ser Val Leu Phe Leu Cys Asp Met Gln Glu Lys Phe Arg His Asn 20 25 30 Ile Ala Tyr Phe Pro Gln Ile Val Ser Val Ala Ala Arg Met Leu 35 40 45 Lys Val Ala Arg Leu Leu Glu Val Pro Val Met Leu Thr Glu Gln 50 55 60 Tyr Pro Gln Gly Leu Gly Pro Thr Val Pro Glu Leu Gly Thr Glu 65 70 75 Gly Leu Arg Pro Leu Ala Lys Thr Cys Phe Ser Met Val Pro Ala 80 85 90 Leu Gln Gln Glu Leu Asp Ser Arg Pro Gln Leu Arg Ser Val Leu 95 100 105 Leu Cys Gly Ile Glu Ala Gln Ala Cys Ile Leu Asn Thr Thr Leu 110 115 120 Asp Leu Leu Asp Arg Gly Leu Gln Val His Val Val Val Asp Ala 125 130 135 Cys Ser Ser Arg Ser Gln Val Asp Arg Leu Val Ala Leu Ala Arg 140 145 150 Met Arg Gln Ser Gly Ala Phe Leu Ser Thr Ser Glu Gly Leu Ile 155 160 165 Leu Gln Leu Val Gly Asp Ala Val His Pro Gln Phe Lys Glu Ile 170 175 180 Gln Lys Leu Ile Lys Glu Pro Ala Pro Asp Ser Gly Leu Leu Gly 185 190 195 Leu Phe Gln Gly Gln Asn Ser Leu Leu His 200 205 419 amino acids amino acid single linear BLADNOT03 1600438 30 Met Asn Lys His Gln Lys Pro Val Leu Thr Gly Gln Arg Phe Lys 5 10 15 Thr Arg Lys Arg Asp Glu Lys Glu Lys Phe Glu Pro Thr Val Phe 20 25 30 Arg Asp Thr Leu Val Gln Gly Leu Asn Glu Ala Gly Asp Asp Leu 35 40 45 Glu Ala Val Ala Lys Phe Leu Asp Ser Thr Gly Ser Arg Leu Asp 50 55 60 Tyr Arg Arg Tyr Ala Asp Thr Leu Phe Asp Ile Leu Val Ala Gly 65 70 75 Ser Met Leu Ala Pro Gly Gly Thr Arg Ile Asp Asp Gly Asp Lys 80 85 90 Thr Lys Met Thr Asn His Cys Val Phe Ser Ala Asn Glu Asp His 95 100 105 Glu Thr Ile Arg Asn Tyr Ala Gln Val Phe Asn Lys Leu Ile Arg 110 115 120 Arg Tyr Lys Tyr Leu Glu Lys Ala Phe Glu Asp Glu Met Lys Lys 125 130 135 Leu Leu Leu Phe Leu Lys Ala Phe Ser Glu Thr Glu Gln Thr Lys 140 145 150 Leu Ala Met Leu Ser Gly Ile Leu Leu Gly Asn Gly Thr Leu Pro 155 160 165 Ala Thr Ile Leu Thr Ser Leu Phe Thr Asp Ser Leu Val Lys Glu 170 175 180 Gly Ile Ala Ala Ser Phe Ala Val Lys Leu Phe Lys Ala Trp Met 185 190 195 Ala Glu Lys Asp Ala Asn Ser Val Thr Ser Ser Leu Arg Lys Ala 200 205 210 Asn Leu Asp Lys Arg Leu Leu Glu Leu Phe Pro Val Asn Arg Gln 215 220 225 Ser Val Asp His Phe Ala Lys Tyr Phe Thr Asp Ala Gly Leu Lys 230 235 240 Glu Leu Ser Asp Phe Leu Arg Val Gln Gln Ser Leu Gly Thr Arg 245 250 255 Lys Glu Leu Gln Lys Glu Leu Gln Glu Arg Leu Ser Gln Glu Cys 260 265 270 Pro Ile Lys Glu Val Val Leu Tyr Val Lys Glu Glu Met Lys Arg 275 280 285 Asn Asp Leu Pro Glu Thr Ala Val Ile Gly Leu Leu Trp Thr Cys 290 295 300 Ile Met Asn Ala Val Glu Trp Asn Lys Lys Glu Glu Leu Val Ala 305 310 315 Glu Gln Ala Leu Lys His Leu Lys Gln Tyr Ala Pro Leu Leu Ala 320 325 330 Val Phe Ser Ser Gln Gly Gln Ser Glu Leu Ile Leu Leu Gln Lys 335 340 345 Val Gln Glu Tyr Cys Tyr Asp Asn Ile His Phe Met Lys Ala Phe 350 355 360 Gln Lys Ile Val Val Leu Phe Tyr Lys Ala Asp Val Leu Ser Glu 365 370 375 Glu Ala Ile Leu Lys Trp Tyr Lys Glu Ala His Val Ala Lys Gly 380 385 390 Lys Ser Val Phe Leu Asp Gln Met Lys Lys Phe Val Glu Trp Leu 395 400 405 Gln Asn Ala Glu Glu Glu Ser Glu Ser Glu Gly Glu Glu Asn 410 415 376 amino acids amino acid single linear BLADNOT03 1600518 31 Met Lys Asp Val Pro Gly Phe Leu Gln Gln Ser Gln Ser Ser Gly 5 10 15 Pro Gly Gln Pro Ala Val Trp His Arg Leu Glu Glu Leu Tyr Thr 20 25 30 Lys Lys Leu Trp His Gln Leu Thr Leu Gln Val Leu Asp Phe Val 35 40 45 Gln Asp Pro Cys Phe Ala Gln Gly Asp Gly Leu Ile Lys Leu Tyr 50 55 60 Glu Asn Phe Ile Ser Glu Phe Glu His Arg Val Asn Pro Leu Ser 65 70 75 Leu Val Glu Ile Ile Leu His Val Val Arg Gln Met Thr Asp Pro 80 85 90 Asn Val Ala Leu Thr Phe Leu Glu Lys Thr Arg Glu Lys Val Lys 95 100 105 Ser Ser Asp Glu Ala Val Ile Leu Cys Lys Thr Ala Ile Gly Ala 110 115 120 Leu Lys Leu Asn Ile Gly Asp Leu Gln Val Thr Lys Glu Thr Ile 125 130 135 Glu Asp Val Glu Glu Met Leu Asn Asn Leu Pro Gly Val Thr Ser 140 145 150 Val His Ser Arg Phe Tyr Asp Leu Ser Ser Lys Tyr Tyr Gln Thr 155 160 165 Ile Gly Asn His Ala Ser Tyr Tyr Lys Asp Ala Leu Arg Phe Leu 170 175 180 Gly Cys Val Asp Ile Lys Asp Leu Pro Val Ser Glu Gln Gln Glu 185 190 195 Arg Ala Phe Thr Leu Gly Leu Ala Gly Leu Leu Gly Glu Gly Val 200 205 210 Phe Asn Phe Gly Glu Leu Leu Met His Pro Val Leu Glu Ser Leu 215 220 225 Arg Asn Thr Asp Arg Gln Trp Leu Ile Asp Thr Leu Tyr Ala Phe 230 235 240 Asn Ser Gly Asn Val Glu Arg Phe Gln Thr Leu Lys Thr Ala Trp 245 250 255 Gly Gln Gln Pro Asp Leu Ala Ala Asn Glu Ala Gln Leu Leu Arg 260 265 270 Lys Ile Gln Leu Leu Cys Leu Met Glu Met Thr Phe Thr Arg Pro 275 280 285 Ala Asn His Arg Gln Leu Thr Phe Glu Glu Ile Ala Lys Ser Ala 290 295 300 Lys Ile Thr Val Asn Glu Val Glu Leu Leu Val Met Lys Ala Leu 305 310 315 Ser Val Gly Leu Val Lys Gly Ser Ile Asp Glu Val Asp Lys Arg 320 325 330 Val His Met Thr Trp Val Gln Pro Arg Val Leu Asp Leu Gln Gln 335 340 345 Ile Lys Gly Met Lys Asp Arg Leu Glu Phe Trp Cys Thr Asp Val 350 355 360 Lys Ser Met Glu Met Leu Val Glu His Gln Ala His Asp Ile Leu 365 370 375 Thr 237 amino acids amino acid single linear BLADNOT03 1602473 32 Met Leu Gly Gly Ser Leu Gly Ser Arg Leu Leu Arg Gly Val Gly 5 10 15 Gly Ser His Gly Arg Phe Gly Ala Arg Gly Val Arg Glu Gly Gly 20 25 30 Ala Ala Met Ala Ala Gly Glu Ser Met Ala Gln Arg Met Val Trp 35 40 45 Val Asp Leu Glu Met Thr Gly Leu Asp Ile Glu Lys Asp Gln Ile 50 55 60 Ile Glu Met Ala Cys Leu Ile Thr Asp Ser Asp Leu Asn Ile Leu 65 70 75 Ala Glu Gly Pro Asn Leu Ile Ile Lys Gln Pro Asp Glu Leu Leu 80 85 90 Asp Ser Met Ser Asp Trp Cys Lys Glu His His Gly Lys Ser Gly 95 100 105 Leu Thr Lys Ala Val Lys Glu Ser Thr Ile Thr Leu Gln Gln Ala 110 115 120 Glu Tyr Glu Phe Leu Ser Phe Val Arg Gln Gln Thr Pro Pro Gly 125 130 135 Leu Cys Pro Leu Ala Gly Asn Ser Val His Glu Asp Lys Lys Phe 140 145 150 Leu Asp Lys Tyr Met Pro Gln Phe Met Lys His Leu His Tyr Arg 155 160 165 Ile Ile Asp Val Ser Thr Val Lys Glu Leu Cys Arg Arg Trp Tyr 170 175 180 Pro Glu Glu Tyr Glu Phe Ala Pro Lys Lys Ala Ala Ser His Arg 185 190 195 Ala Leu Asp Asp Ile Ser Glu Ser Ile Lys Glu Leu Gln Phe Tyr 200 205 210 Arg Asn Asn Ile Phe Lys Lys Lys Ile Asp Glu Lys Lys Arg Lys 215 220 225 Ile Ile Glu Asn Gly Glu Asn Glu Lys Thr Val Ser 230 235 152 amino acids amino acid single linear LUNGNOT15 1605720 33 Met Glu Ala Val Leu Asn Glu Leu Val Ser Val Glu Asp Leu Leu 5 10 15 Lys Phe Glu Lys Lys Phe Gln Ser Glu Lys Ala Ala Gly Ser Val 20 25 30 Ser Lys Ser Thr Gln Phe Glu Tyr Ala Trp Cys Leu Val Arg Ser 35 40 45 Lys Tyr Asn Asp Asp Ile Arg Lys Gly Ile Val Leu Leu Glu Glu 50 55 60 Leu Leu Pro Lys Gly Ser Lys Glu Glu Gln Arg Asp Tyr Val Phe 65 70 75 Tyr Leu Ala Val Gly Asn Tyr Arg Leu Lys Glu Tyr Glu Lys Ala 80 85 90 Leu Lys Tyr Val Arg Gly Leu Leu Gln Thr Glu Pro Gln Asn Asn 95 100 105 Gln Ala Lys Glu Leu Glu Arg Leu Ile Asp Lys Ala Met Lys Lys 110 115 120 Asp Gly Leu Val Gly Met Ala Ile Val Gly Gly Met Ala Leu Gly 125 130 135 Val Ala Gly Leu Ala Gly Leu Ile Gly Leu Ala Val Ser Lys Ser 140 145 150 Lys Phe 179 amino acids amino acid single linear COLNTUT06 1610501 34 Met Pro Ser Lys Ser Leu Val Met Glu Tyr Leu Ala His Pro Ser 5 10 15 Thr Leu Gly Leu Ala Val Gly Val Ala Cys Gly Met Cys Leu Gly 20 25 30 Trp Ser Leu Arg Val Cys Phe Gly Met Leu Pro Lys Ser Lys Thr 35 40 45 Ser Lys Thr His Thr Asp Thr Glu Ser Glu Ala Ser Ile Leu Gly 50 55 60 Asp Ser Gly Glu Tyr Lys Met Ile Leu Val Val Arg Asn Asp Leu 65 70 75 Lys Met Gly Lys Gly Lys Val Ala Ala Gln Cys Ser His Ala Ala 80 85 90 Val Ser Ala Tyr Lys Gln Ile Gln Arg Arg Asn Pro Glu Met Leu 95 100 105 Lys Gln Trp Glu Tyr Cys Gly Gln Pro Lys Val Val Val Lys Ala 110 115 120 Pro Asp Glu Glu Thr Leu Ile Ala Leu Leu Ala His Ala Lys Met 125 130 135 Leu Gly Leu Thr Val Ser Leu Ile Gln Asp Ala Gly Arg Thr Gln 140 145 150 Ile Ala Pro Gly Ser Gln Thr Val Leu Gly Ile Gly Pro Gly Pro 155 160 165 Ala Asp Leu Ile Asp Lys Val Thr Gly His Leu Lys Leu Tyr 170 175 196 amino acids amino acid single linear BLADNOT06 1720770 35 Met Ser Glu Gly Asp Ser Val Gly Glu Ser Val His Gly Lys Pro 5 10 15 Ser Val Val Tyr Arg Phe Phe Thr Arg Leu Gly Gln Ile Tyr Gln 20 25 30 Ser Trp Leu Asp Lys Ser Thr Pro Tyr Thr Ala Val Arg Trp Val 35 40 45 Val Thr Leu Gly Leu Ser Phe Val Tyr Met Ile Arg Val Tyr Leu 50 55 60 Leu Gln Gly Trp Tyr Ile Val Thr Tyr Ala Leu Gly Ile Tyr His 65 70 75 Leu Asn Leu Phe Ile Ala Phe Leu Ser Pro Lys Val Asp Pro Ser 80 85 90 Leu Met Glu Asp Ser Asp Asp Gly Pro Ser Leu Pro Thr Lys Gln 95 100 105 Asn Glu Glu Phe Arg Pro Phe Ile Arg Arg Leu Pro Glu Phe Lys 110 115 120 Phe Trp His Ala Ala Thr Lys Gly Ile Leu Val Ala Met Val Cys 125 130 135 Thr Phe Phe Asp Ala Phe Asn Val Pro Val Phe Trp Pro Ile Leu 140 145 150 Val Met Tyr Phe Ile Met Leu Phe Cys Ile Thr Met Lys Arg Gln 155 160 165 Ile Lys His Met Ile Lys Tyr Arg Tyr Ile Pro Phe Thr His Gly 170 175 180 Lys Arg Arg Tyr Arg Gly Lys Glu Asp Ala Gly Lys Ala Phe Ala 185 190 195 Ser 612 amino acids amino acid single linear BRAINON01 1832295 36 Met Ala Ala Ala Gly Arg Leu Pro Ser Ser Trp Ala Leu Phe Ser 5 10 15 Pro Leu Leu Ala Gly Leu Ala Leu Leu Gly Val Gly Pro Val Pro 20 25 30 Ala Arg Ala Leu His Asn Val Thr Ala Glu Leu Phe Gly Ala Glu 35 40 45 Ala Trp Gly Thr Leu Ala Ala Phe Gly Asp Leu Asn Ser Asp Lys 50 55 60 Gln Thr Asp Leu Phe Val Leu Arg Glu Arg Asn Asp Leu Ile Val 65 70 75 Phe Leu Ala Asp Gln Asn Ala Pro Tyr Phe Lys Pro Lys Val Lys 80 85 90 Val Ser Phe Lys Asn His Ser Ala Leu Ile Thr Ser Val Val Pro 95 100 105 Gly Asp Tyr Asp Gly Asp Ser Gln Met Asp Val Leu Leu Thr Tyr 110 115 120 Leu Pro Lys Asn Tyr Ala Lys Ser Glu Leu Gly Ala Val Ile Phe 125 130 135 Trp Gly Gln Asn Gln Thr Leu Asp Pro Asn Asn Met Thr Ile Leu 140 145 150 Asn Arg Thr Phe Gln Asp Glu Pro Leu Ile Met Asp Phe Asn Gly 155 160 165 Asp Leu Ile Pro Asp Ile Phe Gly Ile Thr Asn Glu Ser Asn Gln 170 175 180 Pro Gln Ile Leu Leu Gly Gly Asn Leu Ser Trp His Pro Ala Leu 185 190 195 Thr Thr Thr Ser Lys Met Arg Ile Pro His Ser His Ala Phe Ile 200 205 210 Asp Leu Thr Glu Asp Phe Thr Ala Asp Leu Phe Leu Thr Thr Leu 215 220 225 Asn Ala Thr Thr Ser Thr Phe Gln Phe Glu Ile Trp Glu Asn Leu 230 235 240 Asp Gly Asn Phe Ser Val Ser Thr Ile Leu Glu Lys Pro Gln Asn 245 250 255 Met Met Val Val Gly Gln Ser Ala Phe Ala Asp Phe Asp Gly Asp 260 265 270 Gly His Met Asp His Leu Leu Pro Gly Cys Glu Asp Lys Asn Cys 275 280 285 Gln Lys Ser Thr Ile Tyr Leu Val Arg Ser Gly Met Lys Gln Trp 290 295 300 Val Pro Val Leu Gln Asp Phe Ser Asn Lys Gly Thr Leu Trp Gly 305 310 315 Phe Val Pro Phe Val Asp Glu Gln Gln Pro Thr Glu Ile Pro Ile 320 325 330 Pro Ile Thr Leu His Ile Gly Asp Tyr Asn Met Asp Gly Tyr Pro 335 340 345 Asp Ala Leu Val Ile Leu Lys Asn Thr Ser Gly Ser Asn Gln Gln 350 355 360 Ala Phe Leu Leu Glu Asn Val Pro Cys Asn Asn Ala Ser Cys Glu 365 370 375 Glu Ala Arg Arg Met Phe Lys Val Tyr Trp Glu Leu Thr Asp Leu 380 385 390 Asn Gln Ile Lys Asp Ala Met Val Ala Thr Phe Phe Asp Ile Tyr 395 400 405 Glu Asp Gly Ile Leu Asp Ile Val Val Leu Ser Lys Gly Tyr Thr 410 415 420 Lys Asn Asp Phe Ala Ile His Thr Leu Lys Asn Asn Phe Glu Ala 425 430 435 Asp Ala Tyr Phe Val Lys Val Ile Val Leu Ser Gly Leu Cys Ser 440 445 450 Asn Asp Cys Pro Arg Lys Ile Thr Pro Phe Gly Val Asn Gln Pro 455 460 465 Gly Pro Tyr Ile Met Tyr Thr Thr Leu Asp Ala Asn Gly Tyr Leu 470 475 480 Lys Asn Gly Ser Ala Gly Gln Leu Ser Gln Ser Ala His Leu Ala 485 490 495 Leu Gln Leu Pro Tyr Asn Val Leu Gly Leu Gly Arg Ser Ala Asn 500 505 510 Phe Leu Asp His Leu Tyr Val Gly Ile Pro Arg Pro Ser Gly Glu 515 520 525 Lys Ser Ile Arg Lys Gln Glu Trp Thr Ala Ile Ile Pro Asn Ser 530 535 540 Gln Leu Ile Val Ile Pro Tyr Pro His Asn Val Pro Arg Ser Trp 545 550 555 Ser Ala Lys Leu Tyr Leu Thr Pro Ser Asn Ile Val Leu Leu Thr 560 565 570 Ala Ile Ala Leu Ile Gly Val Cys Val Phe Ile Leu Ala Ile Ile 575 580 585 Gly Ile Leu His Trp Gln Glu Lys Lys Ala Asp Asp Arg Glu Lys 590 595 600 Arg Gln Glu Ala His Arg Phe His Phe Asp Ala Met 605 610 101 amino acids amino acid single linear CORPNOT02 1990522 37 Met Ala Ala Pro Leu Ser Val Glu Val Glu Phe Gly Gly Gly Ala 5 10 15 Glu Leu Leu Phe Asp Gly Ile Lys Lys His Arg Val Thr Leu Pro 20 25 30 Gly Gln Glu Glu Pro Trp Asp Ile Arg Asn Leu Leu Ile Trp Ile 35 40 45 Lys Lys Asn Leu Leu Lys Glu Arg Pro Glu Leu Phe Ile Gln Gly 50 55 60 Asp Ser Val Arg Pro Gly Ile Leu Val Leu Ile Asn Asp Ala Asp 65 70 75 Trp Glu Leu Leu Gly Glu Leu Asp Tyr Gln Leu Gln Asp Gln Asp 80 85 90 Ser Val Leu Phe Ile Ser Thr Leu His Gly Gly 95 100 132 amino acids amino acid single linear BRAITUT02 2098087 38 Met Ala Lys Asp Ile Leu Gly Glu Ala Gly Leu His Phe Asp Glu 5 10 15 Leu Asn Lys Leu Arg Val Leu Asp Pro Glu Val Thr Gln Gln Thr 20 25 30 Ile Glu Leu Lys Glu Glu Cys Lys Asp Phe Val Asp Lys Ile Gly 35 40 45 Gln Phe Gln Lys Ile Val Gly Gly Leu Ile Glu Leu Val Asp Gln 50 55 60 Leu Ala Lys Glu Ala Glu Asn Glu Lys Met Lys Ala Ile Gly Ala 65 70 75 Arg Asn Leu Leu Lys Ser Ile Ala Lys Gln Arg Glu Ala Gln Gln 80 85 90 Gln Gln Leu Gln Ala Leu Ile Ala Glu Lys Lys Met Gln Leu Glu 95 100 105 Arg Tyr Arg Val Glu Tyr Glu Ala Leu Cys Lys Val Glu Ala Glu 110 115 120 Gln Asn Glu Phe Ile Asp Gln Phe Ile Phe Gln Lys 125 130 188 amino acids amino acid single linear BRAITUT03 2112230 39 Met Ala Asn Ser Gly Cys Lys Asp Val Thr Gly Pro Asp Glu Glu 5 10 15 Ser Phe Leu Tyr Phe Ala Tyr Gly Ser Asn Leu Leu Thr Glu Arg 20 25 30 Ile His Leu Arg Asn Pro Ser Ala Ala Phe Phe Cys Val Ala Arg 35 40 45 Leu Gln Asp Phe Lys Leu Asp Phe Gly Asn Ser Gln Gly Lys Thr 50 55 60 Ser Gln Thr Trp His Gly Gly Ile Ala Thr Ile Phe Gln Ser Pro 65 70 75 Gly Asp Glu Val Trp Gly Val Val Trp Lys Met Asn Lys Ser Asn 80 85 90 Leu Asn Ser Leu Asp Glu Gln Glu Gly Val Lys Ser Gly Met Tyr 95 100 105 Val Val Ile Glu Val Lys Val Ala Thr Gln Glu Gly Lys Glu Ile 110 115 120 Thr Cys Arg Ser Tyr Leu Met Thr Asn Tyr Glu Ser Ala Pro Pro 125 130 135 Ser Pro Gln Tyr Lys Lys Ile Ile Cys Met Gly Ala Lys Glu Asn 140 145 150 Gly Leu Pro Leu Glu Tyr Gln Glu Lys Leu Lys Ala Ile Glu Pro 155 160 165 Asn Asp Tyr Thr Gly Lys Val Ser Glu Glu Ile Glu Asp Ile Ile 170 175 180 Lys Lys Gly Glu Thr Gln Thr Leu 185 86 amino acids amino acid single linear BRSTTUT02 2117050 40 Met Thr Asp Arg Tyr Thr Ile His Ser Gln Leu Glu His Leu Gln 5 10 15 Ser Lys Tyr Ile Gly Thr Gly His Ala Asp Thr Thr Lys Trp Glu 20 25 30 Trp Leu Val Asn Gln His Arg Asp Ser Tyr Cys Ser Tyr Met Gly 35 40 45 His Phe Asp Leu Leu Asn Tyr Phe Ala Ile Ala Glu Asn Glu Ser 50 55 60 Lys Ala Arg Val Arg Phe Asn Leu Met Glu Lys Met Leu Gln Pro 65 70 75 Cys Gly Pro Pro Ala Asp Lys Pro Glu Glu Asn 80 85 222 amino acids amino acid single linear SININOT01 2184712 41 Met Ser Gly Leu Gly Arg Leu Phe Gly Lys Gly Lys Lys Glu Lys 5 10 15 Gly Pro Thr Pro Glu Glu Ala Ile Gln Lys Leu Lys Glu Thr Glu 20 25 30 Lys Ile Leu Ile Lys Lys Gln Glu Phe Leu Glu Gln Lys Ile Gln 35 40 45 Gln Glu Leu Gln Thr Ala Lys Lys Tyr Gly Thr Lys Asn Lys Arg 50 55 60 Ala Ala Leu Gln Ala Leu Arg Arg Lys Lys Arg Phe Glu Gln Gln 65 70 75 Leu Ala Gln Thr Asp Gly Thr Leu Ser Thr Leu Glu Phe Gln Arg 80 85 90 Glu Ala Ile Glu Asn Ala Thr Thr Asn Ala Glu Val Leu Arg Thr 95 100 105 Met Glu Leu Ala Ala Gln Ser Met Lys Lys Ala Tyr Gln Asp Met 110 115 120 Asp Ile Asp Lys Val Asp Glu Leu Met Thr Asp Ile Thr Glu Gln 125 130 135 Gln Glu Val Ala Gln Gln Ile Ser Asp Ala Ile Ser Arg Pro Met 140 145 150 Gly Phe Gly Asp Asp Val Asp Glu Asp Glu Leu Leu Glu Glu Leu 155 160 165 Glu Glu Leu Glu Gln Glu Glu Leu Ala Gln Glu Leu Leu Asn Val 170 175 180 Gly Asp Lys Glu Glu Glu Pro Ser Val Lys Leu Pro Ser Val Pro 185 190 195 Ser Thr His Leu Pro Ala Gly Pro Ala Pro Lys Val Asp Glu Asp 200 205 210 Glu Glu Ala Leu Lys Gln Leu Ala Glu Trp Val Ser 215 220 300 amino acids amino acid single linear BRAINON01 2290475 42 Met Ser Gly Ser Asn Gly Ser Lys Glu Asn Ser His Asn Lys Ala 5 10 15 Arg Thr Ser Pro Tyr Pro Gly Ser Lys Val Glu Arg Ser Gln Val 20 25 30 Pro Asn Glu Lys Val Gly Trp Leu Val Glu Trp Gln Asp Tyr Lys 35 40 45 Pro Val Glu Tyr Thr Ala Val Ser Val Leu Ala Gly Pro Arg Trp 50 55 60 Ala Asp Pro Gln Ile Ser Glu Ser Asn Phe Ser Pro Lys Phe Asn 65 70 75 Glu Lys Asp Gly His Val Glu Arg Lys Ser Lys Asn Gly Leu Tyr 80 85 90 Glu Ile Glu Asn Gly Arg Pro Arg Asn Pro Ala Gly Arg Thr Gly 95 100 105 Leu Val Gly Arg Gly Leu Leu Gly Arg Trp Gly Pro Asn His Ala 110 115 120 Ala Asp Pro Ile Ile Thr Arg Trp Lys Arg Asp Ser Ser Gly Asn 125 130 135 Lys Ile Met His Pro Val Ser Gly Lys His Ile Leu Gln Phe Val 140 145 150 Ala Ile Lys Arg Lys Asp Cys Gly Glu Trp Ala Ile Pro Gly Gly 155 160 165 Met Val Asp Pro Gly Glu Lys Ile Ser Ala Thr Leu Lys Arg Glu 170 175 180 Phe Gly Glu Glu Ala Leu Asn Ser Leu Gln Lys Thr Ser Ala Glu 185 190 195 Lys Arg Glu Ile Glu Glu Lys Leu His Lys Leu Phe Ser Gln Asp 200 205 210 His Leu Val Ile Tyr Lys Gly Tyr Val Asp Asp Pro Arg Asn Thr 215 220 225 Asp Asn Ala Trp Met Glu Thr Glu Ala Val Asn Tyr His Asp Glu 230 235 240 Thr Gly Glu Ile Met Asp Asn Leu Met Leu Glu Ala Gly Asp Asp 245 250 255 Ala Gly Lys Val Lys Trp Val Asp Ile Asn Asp Lys Leu Lys Leu 260 265 270 Tyr Ala Ser His Ser Gln Phe Ile Lys Leu Val Ala Glu Lys Arg 275 280 285 Asp Ala His Trp Ser Glu Asp Ser Glu Ala Asp Cys His Ala Leu 290 295 300 112 amino acids amino acid single linear LUNGNOT20 2353452 43 Met Glu Ala Tyr Glu Gln Val Gln Lys Gly Pro Leu Lys Leu Lys 5 10 15 Gly Val Ala Glu Leu Gly Val Thr Lys Arg Lys Lys Lys Lys Lys 20 25 30 Asp Lys Asp Lys Ala Lys Leu Leu Glu Ala Met Gly Thr Ser Lys 35 40 45 Lys Asn Glu Glu Glu Lys Arg Arg Gly Leu Asp Lys Arg Thr Pro 50 55 60 Ala Gln Ala Ala Phe Glu Lys Met Gln Glu Lys Arg Gln Met Glu 65 70 75 Arg Ile Leu Lys Lys Ala Ser Lys Thr His Lys Gln Arg Val Glu 80 85 90 Asp Phe Asn Arg His Leu Asp Thr Leu Thr Glu His Tyr Asp Ile 95 100 105 Pro Lys Val Ser Trp Thr Lys 110 251 amino acids amino acid single linear THP1NOT03 2469611 44 Met Ser Asp Ile Gly Asp Trp Phe Arg Ser Ile Pro Ala Ile Thr 5 10 15 Arg Tyr Trp Phe Ala Ala Thr Val Ala Val Pro Leu Val Gly Lys 20 25 30 Leu Gly Leu Ile Ser Pro Ala Tyr Leu Phe Leu Trp Pro Glu Ala 35 40 45 Phe Leu Tyr Arg Phe Gln Ile Trp Arg Pro Ile Thr Ala Thr Phe 50 55 60 Tyr Phe Pro Val Gly Pro Gly Thr Gly Phe Leu Tyr Leu Val Asn 65 70 75 Leu Tyr Phe Leu Tyr Gln Tyr Ser Thr Arg Leu Glu Thr Gly Ala 80 85 90 Phe Asp Gly Arg Pro Ala Asp Tyr Leu Phe Met Leu Leu Phe Asn 95 100 105 Trp Ile Cys Ile Val Ile Thr Gly Leu Ala Met Asp Met Gln Leu 110 115 120 Leu Met Ile Pro Leu Ile Met Ser Val Leu Tyr Val Trp Ala Gln 125 130 135 Leu Asn Arg Asp Met Ile Val Ser Phe Trp Phe Gly Thr Arg Phe 140 145 150 Lys Ala Cys Tyr Leu Pro Trp Val Ile Leu Gly Phe Asn Tyr Ile 155 160 165 Ile Gly Gly Ser Val Ile Asn Glu Leu Ile Gly Asn Leu Val Gly 170 175 180 His Leu Tyr Phe Phe Leu Met Phe Arg Tyr Pro Met Asp Leu Gly 185 190 195 Gly Arg Asn Phe Leu Ser Thr Pro Gln Phe Leu Tyr Arg Trp Leu 200 205 210 Pro Ser Arg Arg Gly Gly Val Ser Gly Phe Gly Val Pro Pro Ala 215 220 225 Ser Met Arg Arg Ala Ala Asp Gln Asn Gly Gly Gly Gly Arg His 230 235 240 Asn Trp Gly Gln Gly Phe Arg Leu Gly Asp Gln 245 250 811 amino acids amino acid single linear LIVRTUT04 2515476 45 Met Pro Leu Ser Ser Pro Asn Ala Ala Ala Thr Ala Ser Asp Met 5 10 15 Asp Lys Asn Ser Gly Ser Asn Ser Ser Ser Ala Ser Ser Gly Ser 20 25 30 Ser Lys Gly Gln Gln Pro Pro Arg Ser Ala Ser Ala Gly Pro Ala 35 40 45 Gly Glu Ser Lys Pro Lys Ser Asp Gly Lys Asn Ser Ser Gly Ser 50 55 60 Lys Arg Tyr Asn Arg Lys Arg Glu Leu Ser Tyr Pro Lys Asn Glu 65 70 75 Ser Phe Asn Asn Gln Ser Arg Arg Ser Ser Ser Gln Lys Ser Lys 80 85 90 Thr Phe Asn Lys Met Pro Pro Gln Arg Gly Gly Gly Ser Ser Lys 95 100 105 Leu Phe Ser Ser Ser Phe Asn Gly Gly Arg Arg Asp Glu Val Ala 110 115 120 Glu Ala Gln Arg Ala Glu Phe Ser Pro Ala Gln Phe Ser Gly Pro 125 130 135 Lys Lys Ile Asn Leu Asn His Leu Leu Asn Phe Thr Phe Glu Pro 140 145 150 Arg Gly Gln Thr Gly His Phe Glu Gly Ser Gly His Gly Ser Trp 155 160 165 Gly Lys Arg Asn Lys Trp Gly His Lys Pro Phe Asn Lys Glu Leu 170 175 180 Phe Leu Gln Ala Asn Cys Gln Phe Val Val Ser Glu Asp Gln Asp 185 190 195 Tyr Thr Ala His Phe Ala Asp Pro Asp Thr Leu Val Asn Trp Asp 200 205 210 Phe Val Glu Gln Val Arg Ile Cys Ser His Glu Val Pro Ser Cys 215 220 225 Pro Ile Cys Leu Tyr Pro Pro Thr Ala Ala Lys Ile Thr Arg Cys 230 235 240 Gly His Ile Phe Cys Trp Ala Cys Ile Leu His Tyr Leu Ser Leu 245 250 255 Ser Glu Lys Thr Trp Ser Lys Cys Pro Ile Cys Tyr Ser Ser Val 260 265 270 His Lys Lys Asp Leu Lys Ser Val Val Ala Thr Glu Ser His Gln 275 280 285 Tyr Val Val Gly Asp Thr Ile Thr Met Gln Leu Met Lys Arg Glu 290 295 300 Lys Gly Val Leu Val Ala Leu Pro Lys Ser Lys Trp Met Asn Val 305 310 315 Asp His Pro Ile His Leu Gly Asp Glu Gln His Ser Gln Tyr Ser 320 325 330 Lys Leu Leu Leu Ala Ser Lys Glu Gln Val Leu His Arg Val Val 335 340 345 Leu Glu Glu Lys Val Ala Leu Glu Gln Gln Leu Ala Glu Glu Lys 350 355 360 His Thr Pro Glu Ser Cys Phe Ile Glu Ala Ala Ile Gln Glu Leu 365 370 375 Lys Thr Arg Glu Glu Ala Leu Ser Gly Leu Ala Gly Ser Arg Arg 380 385 390 Glu Val Thr Gly Val Val Ala Ala Leu Glu Gln Leu Val Leu Met 395 400 405 Ala Pro Leu Ala Lys Glu Ser Val Phe Gln Pro Arg Lys Gly Val 410 415 420 Leu Glu Tyr Leu Ser Ala Phe Asp Glu Glu Thr Thr Glu Val Cys 425 430 435 Ser Leu Asp Thr Pro Ser Arg Pro Leu Ala Leu Pro Leu Val Glu 440 445 450 Glu Glu Glu Ala Val Ser Glu Pro Glu Pro Glu Gly Leu Pro Glu 455 460 465 Ala Cys Asp Asp Leu Glu Leu Ala Asp Asp Asn Leu Lys Glu Gly 470 475 480 Thr Ile Cys Thr Glu Ser Ser Gln Gln Glu Pro Ile Thr Lys Ser 485 490 495 Gly Phe Thr Arg Leu Ser Ser Ser Pro Cys Tyr Tyr Phe Tyr Gln 500 505 510 Ala Glu Asp Gly Gln His Met Phe Leu His Pro Val Asn Val Arg 515 520 525 Cys Leu Val Arg Glu Tyr Gly Ser Leu Glu Arg Ser Pro Glu Lys 530 535 540 Ile Ser Ala Thr Val Val Glu Ile Ala Gly Tyr Ser Met Ser Glu 545 550 555 Asp Val Arg Gln Arg His Arg Tyr Leu Ser His Leu Pro Leu Thr 560 565 570 Cys Glu Phe Ser Ile Cys Glu Leu Ala Leu Gln Pro Pro Val Val 575 580 585 Ser Lys Glu Thr Leu Glu Met Phe Ser Asp Asp Ile Glu Lys Arg 590 595 600 Lys Arg Gln Arg Gln Lys Lys Ala Arg Glu Glu Arg Arg Arg Glu 605 610 615 Arg Arg Ile Glu Ile Glu Glu Asn Lys Lys Gln Gly Lys Tyr Pro 620 625 630 Glu Val His Ile Pro Leu Glu Asn Leu Gln Gln Phe Pro Ala Phe 635 640 645 Asn Ser Tyr Thr Cys Ser Ser Asp Ser Ala Leu Gly Pro Thr Ser 650 655 660 Thr Glu Gly His Gly Ala Leu Ser Ile Ser Pro Leu Ser Arg Ser 665 670 675 Pro Gly Ser His Ala Asp Phe Leu Leu Thr Pro Leu Ser Pro Thr 680 685 690 Ala Ser Gln Gly Ser Pro Ser Phe Cys Val Gly Ser Leu Glu Glu 695 700 705 Asp Ser Pro Phe Pro Ser Phe Ala Gln Met Leu Arg Val Gly Lys 710 715 720 Ala Lys Ala Asp Val Trp Pro Lys Thr Ala Pro Lys Lys Asp Glu 725 730 735 Asn Ser Leu Val Pro Pro Ala Pro Val Asp Ser Asp Gly Glu Ser 740 745 750 Asp Asn Ser Asp Arg Val Pro Val Pro Ser Phe Gln Asn Ser Phe 755 760 765 Ser Gln Ala Ile Glu Ala Ala Phe Met Lys Leu Asp Thr Pro Ala 770 775 780 Thr Ser Asp Pro Leu Ser Glu Glu Lys Gly Gly Lys Lys Arg Lys 785 790 795 Lys Gln Lys Gln Lys Leu Leu Phe Ser Thr Ser Val Val His Thr 800 805 810 Lys 352 amino acids amino acid single linear THP1AZS08 2754573 46 Met His Val Val Ala Pro Ala Ser Leu Arg Leu Gly Thr Gly Thr 5 10 15 Asn Leu Pro Pro Ser Pro Thr Cys Leu Thr Lys Leu Ala Leu Pro 20 25 30 Pro Ala Ala Glu Pro Ser Leu Leu Ala Met Ser Gln Ser Arg His 35 40 45 Arg Ala Glu Ala Pro Pro Leu Glu Arg Glu Asp Ser Gly Thr Phe 50 55 60 Ser Leu Gly Lys Met Ile Thr Ala Lys Pro Gly Lys Thr Pro Ile 65 70 75 Gln Val Leu His Glu Tyr Gly Met Lys Thr Lys Asn Ile Pro Val 80 85 90 Tyr Glu Cys Glu Arg Ser Asp Val Gln Ile His Val Pro Thr Phe 95 100 105 Thr Phe Arg Val Thr Val Gly Asp Ile Thr Cys Thr Gly Glu Gly 110 115 120 Thr Ser Lys Lys Leu Ala Lys His Arg Ala Ala Glu Ala Ala Ile 125 130 135 Asn Ile Leu Lys Ala Asn Ala Ser Ile Cys Phe Ala Val Pro Asp 140 145 150 Pro Leu Met Pro Asp Pro Ser Lys Gln Pro Lys Asn Gln Leu Asn 155 160 165 Pro Ile Gly Ser Leu Gln Glu Leu Ala Ile His His Gly Trp Arg 170 175 180 Leu Pro Glu Tyr Thr Leu Ser Gln Glu Gly Gly Pro Ala His Lys 185 190 195 Arg Glu Tyr Thr Thr Ile Cys Arg Leu Glu Ser Phe Met Glu Thr 200 205 210 Gly Lys Gly Ala Ser Lys Lys Gln Ala Lys Arg Asn Ala Ala Glu 215 220 225 Lys Phe Leu Ala Lys Phe Ser Asn Ile Ser Pro Glu Asn His Ile 230 235 240 Ser Leu Thr Asn Val Val Gly His Ser Leu Gly Cys Thr Trp His 245 250 255 Ser Leu Arg Asn Ser Pro Gly Glu Lys Ile Asn Leu Leu Lys Arg 260 265 270 Ser Leu Leu Ser Ile Pro Asn Thr Asp Tyr Ile Gln Leu Leu Ser 275 280 285 Glu Ile Ala Lys Glu Gln Gly Phe Asn Ile Thr Tyr Leu Asp Ile 290 295 300 Asp Glu Leu Ser Ala Asn Gly Gln Tyr Gln Cys Leu Ala Glu Leu 305 310 315 Ser Thr Ser Pro Ile Thr Val Cys His Gly Ser Gly Ile Ser Cys 320 325 330 Gly Asn Ala Gln Ser Asp Ala Ala His Asn Ala Leu Gln Tyr Leu 335 340 345 Lys Ile Ile Ala Glu Arg Lys 350 432 amino acids amino acid single linear TLYMNOT04 2926777 47 Met Ile Ser Ala Ala Gln Leu Leu Asp Glu Leu Met Gly Arg Asp 5 10 15 Arg Asn Leu Ala Pro Asp Glu Lys Arg Thr Asn Val Arg Trp Asp 20 25 30 His Glu Ser Val Cys Lys Tyr Tyr Leu Cys Gly Phe Cys Pro Ala 35 40 45 Glu Leu Phe Thr Asn Thr Arg Ser Asp Leu Gly Pro Cys Glu Lys 50 55 60 Ile His Asp Glu Asn Leu Arg Lys Gln Tyr Glu Lys Ser Ser Arg 65 70 75 Phe Met Lys Val Gly Tyr Glu Arg Asp Phe Leu Arg Tyr Leu Gln 80 85 90 Ser Leu Leu Ala Glu Val Glu Arg Arg Ile Arg Arg Gly His Ala 95 100 105 Arg Leu Ala Leu Ser Gln Asn Gln Gln Ser Ser Gly Ala Ala Gly 110 115 120 Pro Thr Gly Lys Asn Glu Glu Lys Ile Gln Val Leu Thr Asp Lys 125 130 135 Ile Asp Val Leu Leu Gln Gln Ile Glu Glu Leu Gly Ser Glu Gly 140 145 150 Lys Val Glu Glu Ala Gln Gly Met Met Lys Leu Val Glu Gln Leu 155 160 165 Lys Glu Glu Arg Glu Leu Leu Arg Ser Thr Thr Ser Thr Ile Glu 170 175 180 Ser Phe Ala Ala Gln Glu Lys Gln Met Glu Val Cys Glu Val Cys 185 190 195 Gly Ala Phe Leu Ile Val Gly Asp Ala Gln Ser Arg Val Asp Asp 200 205 210 His Leu Met Gly Lys Gln His Met Gly Tyr Ala Lys Ile Lys Ala 215 220 225 Thr Val Glu Glu Leu Lys Glu Lys Leu Arg Lys Arg Thr Glu Glu 230 235 240 Pro Asp Arg Asp Glu Arg Leu Lys Lys Glu Lys Gln Glu Arg Glu 245 250 255 Glu Arg Glu Lys Glu Arg Glu Arg Glu Arg Glu Glu Arg Glu Arg 260 265 270 Lys Arg Arg Arg Glu Glu Glu Glu Arg Glu Lys Glu Arg Ala Arg 275 280 285 Asp Arg Glu Arg Arg Lys Arg Ser Arg Ser Arg Ser Arg His Ser 290 295 300 Ser Arg Thr Ser Asp Arg Arg Cys Ser Arg Ser Arg Asp His Lys 305 310 315 Arg Ser Arg Ser Arg Glu Arg Arg Arg Thr Arg Ser Arg Asp Arg 320 325 330 Arg Arg Ser Arg Ser His Asp Arg Ser Glu Arg Lys His Arg Ser 335 340 345 Arg Ser Arg Asp Arg Arg Arg Ser Lys Ser Arg Asp Arg Lys Ser 350 355 360 Tyr Lys His Arg Ser Lys Ser Arg Asp Arg Glu Gln Asp Arg Lys 365 370 375 Ser Lys Glu Lys Glu Lys Arg Gly Ser Asp Asp Lys Lys Ser Ser 380 385 390 Val Lys Ser Gly Ser Arg Glu Lys Gln Ser Glu Asp Thr Asn Thr 395 400 405 Glu Ser Lys Glu Ser Asp Thr Lys Asn Glu Val Asn Gly Thr Ser 410 415 420 Glu Asp Ile Lys Ser Glu Gly Asp Thr Gln Ser Asn 425 430 180 amino acids amino acid single linear TESTNOT07 3217567 48 Met Ala Ala Ala Glu Glu Glu Asp Gly Gly Pro Glu Gly Pro Asn 5 10 15 Arg Glu Arg Gly Gly Ala Gly Ala Thr Phe Glu Cys Asn Ile Cys 20 25 30 Leu Glu Thr Ala Arg Glu Ala Val Val Ser Val Cys Gly His Leu 35 40 45 Tyr Cys Trp Pro Cys Leu His Gln Trp Leu Glu Thr Arg Pro Glu 50 55 60 Arg Gln Glu Cys Pro Val Cys Lys Ala Gly Ile Ser Arg Glu Lys 65 70 75 Val Val Pro Leu Tyr Gly Arg Gly Ser Gln Lys Pro Gln Asp Pro 80 85 90 Arg Leu Lys Thr Pro Pro Arg Pro Gln Gly Gln Arg Pro Ala Pro 95 100 105 Glu Ser Arg Gly Gly Phe Gln Pro Phe Gly Asp Thr Gly Gly Phe 110 115 120 His Phe Ser Phe Gly Val Gly Ala Phe Pro Phe Gly Phe Phe Thr 125 130 135 Thr Val Phe Asn Ala His Glu Pro Phe Arg Arg Gly Thr Gly Val 140 145 150 Asp Leu Gly Gln Gly His Pro Ala Ser Ser Trp Gln Asp Ser Leu 155 160 165 Phe Leu Phe Leu Ala Ile Phe Phe Phe Phe Trp Leu Leu Ser Ile 170 175 180 137 amino acids amino acid single linear SPLNNOT10 3339274 49 Met Ser Ser Leu Ile Arg Arg Val Ile Ser Thr Ala Lys Ala Pro 5 10 15 Gly Ala Ile Gly Pro Tyr Ser Gln Ala Val Leu Val Asp Arg Thr 20 25 30 Ile Tyr Ile Ser Gly Gln Ile Gly Met Asp Pro Ser Ser Gly Gln 35 40 45 Leu Val Ser Gly Gly Val Ala Glu Glu Ala Lys Gln Ala Leu Lys 50 55 60 Asn Met Gly Glu Ile Leu Lys Ala Ala Gly Cys Asp Phe Thr Asn 65 70 75 Val Val Lys Thr Thr Val Leu Leu Ala Asp Ile Asn Asp Phe Asn 80 85 90 Thr Val Asn Glu Ile Tyr Lys Gln Tyr Phe Lys Ser Asn Phe Pro 95 100 105 Ala Arg Ala Ala Tyr Gln Val Ala Ala Leu Pro Lys Gly Ser Arg 110 115 120 Ile Glu Ile Glu Ala Val Ala Ile Gln Gly Pro Leu Thr Thr Ala 125 130 135 Ser Leu 1600 base pairs nucleic acid single linear U937NOT01 133 50 CCCGGGGGCC CGGGCGGCAG GGCAAGCAGC GCGGCCTCGG CCTATGCGAC CGGTGGCGCC 60 GGCGCGGCTT CTGCCTGGAG AGGATTCAAG ATGACCAACG AAGAACCTCT TCCCAAGAA 120 GTTCGATTGA GTGAAACAGA CTTCAAAGTT ATGGCAAGAG ATGAGTTAAT TCTAAGATG 180 AAACAATATG AAGCATATGT ACAAGCTTTG GAGGGCAAGT ACACAGATCT TAACTCTAA 240 GATGTAACTG GCCTAAGAGA GTCTGAAGAA AAACTAAAGC AACAACAGCA GGAGTCTGC 300 CGCAGGGAAA ACATCCTTGT AATGCGACTA GCAACCAAGG AACAAGAGAT GCAAGAGTG 360 ACTACTCAAA TCCAGTACCT CAAGCAAGTC CAGCAGCCGA GCGTTGCCCA ACTGAGATC 420 ACAATGGTAG ACCCAGCGAT CAACTTGTTT TTCCTAAAAA TGAAAGGTGA ACTGGAACA 480 ACTAAAGACA AACTGGAACA AGCCCAAAAT GAACTGAGTG CCTGGAAGTT TACGCCTGA 540 AGGTAAACAA ATCATACTCC CCAGTCAAGA CTTCCCTGAC AGTCCCACTA CGAGAAAGC 600 GTGGTGGGAC AGCCAAGTAC TCGTTTCCAC ACCAAGACTC AGACTTTTTG AGCCAAAAA 660 AAGCCACATT CTTACACTGT CCAGCTTGTA ATGGTTAATG TAAAACTTAC CAGATGAAC 720 TTGTGTTTCA GCTTTTTTCT TTTCCCCTTC CCCTTGCTTC AGAGGCCTGA TGGCGTCGG 780 CTATTCCGAA GAAGTGGCCA CCTCCGAAAA ATTCCCCTTC TAGAACATGT AGACACTTG 840 GAAATGTTTC TGTTTGAAGA AAATAGAGGG AGAAACAGAA GTCTTAAGTC TGTGGCACA 900 TGTGTCTTCA GACAGTTTGA AGGAATGAAA ACCTAGAGAT TTTAAATCAT GAATTGAAC 960 TGTAAAATTC CAGTAAAATG TAAAAACGGA ATATGCATCG CTCTTAACCT TGAGCATA 1020 GACTTAGAGA CACTGTGTAT CAGTTTTGCC AATAAGACTG TGGACTTCAT GATTGTTG 1080 GAACTTCTGG GTCAAAACTC AAATGAGGTG AATTTTGCCT TTAAAGGGTT TATTTGCT 1140 GAACCAACTT TCAATAGTCA TGAGAGAATC AAATAATAGA TGTCCGTACA AGTAGCGC 1200 ATATTTAACC ATTTAGTTTG GGGCTCTATA TTACTTGCTT GAGCCTTAAT CAATGTGG 1260 TTATTCAATG GTTTGTTCTT TGAATGGTTG CAAAAACTGT AGATAATCTT ACTGAGGA 1320 GTACAAACAT GAAGGTGTGG TATCAAACTT CAGGTTGAAA CTGTTTGAAG CATTATAA 1380 ATTCATTTCA CAACTAGATT GTATAAGGAT ATTAGCTGTG ATGAGACTCA CTGCATTA 1440 TTTTTTAGTG AATTTTATGA AATCCCCGTT CCATTCAACA GGCACATGTT TAAAAGAG 1500 TTGTCGTTGG TGTTAATGGG GGAATGTGTT CCTTCATTGT ATTTGGGCCT TTTGTATT 1560 ACTCTTGATA TTAAATTAAA TGTGCCTTGA AAAAAAAAAA 1600 1033 base pairs nucleic acid single linear U937NOT01 1762 51 GCCGCCGGGA GCCGGTGCGG CTGTGAGGGG CCGCGTCTCG CAGCAGCCGC CCGGACCGGC 60 ATGGTGTTGG GCGCCGGGCC CGCCTCGCCT GTCTCGGGGA GCCCAGGGTA AAGGCAGCA 120 TAATGCTAAC GCTAGCAAGT AAACTGAAGC GTGACGATGG TCTCAAAGGG TCCCGGACG 180 CAGCCACAGC GTCCGACTCG ACTCGGAGGG TTTCTGTGAG AGACAAATTG CTTGTTAAA 240 AGGTTGCAGA ACTTGAAGCT AATTTACCTT GTACATGTAA AGTGCATTTT CCTGATCCA 300 ACAAGCTTCA TTGTTTTCAG CTAACAGTAA CCCCAGATGA GGGTTACTAC CAGGGTGGA 360 AATTTCAGTT TGAAACTGAA GTTCCCGATG CGTACAACAT GGTGCCTCCC AAAGTGAAA 420 GCCTGACCAA GATCTGGCAC CCCAACATCA CAGAGACAGG GGAAATATGT CTGAGTTTA 480 TGAGAGAACA TTCAATTGAT GGCACTGGCT GGGCTCCCAC AAGAACATTA AAGGATGTC 540 TTTGGGGATT AAACTCTTTG TTTACTGATC TTTTGAATTT TGATGATCCA CTGAATATT 600 AAGCTGCAGA ACATCATTTG CGGGACAAGG AGGACTTCCG GAATAAAGTG GATGACTAC 660 TCAAACGTTA TGCCAGATGA TAAAAGGGGA CGATTGCAGG CCCATGGACT GTGTTACAG 720 TTGTCTCTAA CATGAAACAG CAAGAGGTAG CCCCCTCTCC CGTCCTCATG CTCCCTCTC 780 GTCCCCTGGA TTGCCCCAGT CCTGTGACCA TGTTGCCCTG AAGAAGACCA TCTTCATGA 840 TGCTCATTGT AGATGGAGAA TTCAACATAA ATACAGCAAG AAAATGTGTT TGGGCTTCT 900 AAGAGTTGTC TGCTTACCTT AACATGTTTA CTTTTTTGAA CTTGTACTGT ATAGGCTGT 960 GGTGAAATTC TTAAGAAGTT GTAATGAACT CAAAATTGAG GCCAGAGCTT GCTTTCCC 1020 TTCCCAAACA AAA 1033 1837 base pairs nucleic acid single linear U937NOT01 1847 52 TCGGAGAGGC ATCTGGGTTC GGACTGGGGC CGCCATGGGG AAAGTGAATG TGGCCAAGTT 60 GCGTTACATG AGCCGAGATG ACTTCAGGGT CTTGACCGCG GTTGAAATGG GCATGAAGA 120 CCATGAAATT GTTCCCGGCA GTTTGATTGC TTCTATAGCC AGCCTTAAAC ATGGTGGCT 180 TAATAAAGTT TTAAGAGAAT TAGTGAAACA TAAACTCATA GCTTGGGAGC GTACCAAAA 240 TGTCCAGGGC TATCGGTTGA CAAATGCAGG ATATGATTAC CTAGCTTTGA AAACACTTT 300 TTCTAGGCAA GTAGTTGAGT CTGTTGGAAA CCAGATGGGT GTTGGCAAAG AATCAGATA 360 TTACATTGTT GCAAATGAAG AAGGACAACA ATTTGCATTA AAGCTTCACA GACTAGGAA 420 AACCTCGTTT CGAAATTTGA AAAACAAACG CGATTATCAT AAACATAGGC ACAATGTGT 480 TTGGCTTTAT TTATCTCGTC TCTCTGCCAT GAAGGAATTT GCCTATATGA AGGCATTGT 540 TGAGAGGAAA TTTCCAGTTC CAAAGCCAAT TGATTACAAT CGTCATGCAG TGGTCATGG 600 ACTCATAAAT GGTTATCCAC TATGTCAGAT ACACCATGTT GAAGATCCTG CATCAGTAT 660 TGATGAAGCT ATGGAACTAA TTGTCAAACT TGCAAATCAT GGGCTGATTC ATGGAGATT 720 TAATGAATTT AATCTCATTT TGGATGAAAG TGACCATATC ACCATGATTG ATTTTCCAC 780 GATGGTTTCA ACTTCTCATC CCAATGCTGA GTGGTATTTT GACAGAGATG TTAAATGCA 840 TAAAGATTTC TTTATGAAAC GTTTCAGCTA CGAAAGTGAG CTTTTTCCAA CTTTTAAGG 900 TATCAGGAGA GAAGACACTC TTGATGTGGA GGTTTCTGCC AGTGGCTACA CAAAGGAAA 960 GCAGGCAGAT GATGAACTGC TTCATCCATT AGGTCCAGAT GATAAAAATA TTGAAACA 1020 AGAGGGATCT GAATTCTCAT TTTCAGATGG AGAAGTGGCA GAAAAAGCAG AGGTTTAC 1080 GTCAGAAAAT GAAAGTGAAC GGAACTGTCT AGAAGAATCA GAGGGCTGCT ATTGCAGA 1140 ATCTGGAGAC CCTGAACAAA TAAAGGAAGA CAGTTTATCA GAAGAGAGTG CTGATGCA 1200 GAGTTTTGAA ATGACTGAAT TCAATCAAGC TTTAGAAGAA ATAAAAGGGC AGGTTGTT 1260 AAACAACTCT GTAACTGAAT TTTCTGAGGA GAAAAACAGA ACTGAAAATT ACAACAGG 1320 AGATGGTCAG AGAGTTCAAG GAGGAGTCCC TGCTGGCTCT GACGAGTATG AAGATGAA 1380 CCCTCATCTA ATTGCCTTGT CGTCATTAAA TAGAGAATTC AGGCCTTTCA GAGATGAA 1440 AAATGTGGGA GCTATGAATC AGTATAGAAC AAGAACTCTG AGTATCACTT CTTCAGGC 1500 TGCTGTAAGC TGTTCAACAA TTCCTCCAGA ACTGGTGAAA CAGAAGGTGA AACGTCAG 1560 GACAAAACAG CAAAAATCAG CTGTCAGACG TCGATTGCAG AAAGGAGAAG CAAATATA 1620 TACCAAGCAA CGTAGGGAAA ACATGCAAAA TATCAAATCA AGTTTGGAAG CAGCTAGC 1680 TTGGGGAGAA TAATATATTT AGGATCTTGG ATATGTTTAA TATATTTTTT AAAGTTAC 1740 TAATTCCTTT TTGAGCCCTC ATTTGTCTTT TTTGAGCCAA GGCTATCATA TATTAATA 1800 TAAACCCTCC TTTCATCTAT AAAAAAAAAA AAAAAAA 1837 2031 base pairs nucleic acid single linear HMC1NOT01 9337 53 CGCGCCGGGA CTCTGCCCAC TTCCACCAGA GACACATTGA GAAGGAGGAA ACTATGGCCT 60 CCAGGCTTCC GACGGCCTGG TCCTGTGAAC CAGTGACCTT TGAAGATGTA ACACTGGGT 120 TTACCCCGGA AGAGTGGGGA CTGCTGGACC TCAAACAGAA GTCCCTGTAC AGGGAAGTG 180 TGCTGGAGAA CTACAGGAAC CTGGTCTCAG TGGAACATCA GCTTTCCAAA CCAGATGTG 240 TATCTCAGTT AGAGGAGGCA GAAGATTTCT GGCCAGTGGA GAGAGGAATT CCTCAAGAC 300 CCATTCCAGA GTATCCTGAG CTCCAGCTGG ACCCTAAATT GGATCCTCTT CCTGCTGAG 360 GTCCCCTAAT GAACATTGAG GTTGTTGAGG TCCTCACACT GAACCAGGAG GTGGCTGGT 420 CCCGGAATGC CCAGATCCAG GCCCTATATG CTGAAGATGG AAGCCTGAGT GCAGATGCC 480 CCAGTGAGCA GGTCCAACAG CAGGGCAAGC ATCCAGGTGA CCCTGAGGCC GCGCGCCAG 540 GGTTCCGGCA GTTCCGTTAT AAGGACATGA CAGGTCCCCG GGAGGCCCTG GACCAGCTC 600 GAGAGCTGTG TCACCAGTGG CTACAGCCTA AGGCACGCTC CAAGGAGCAG ATCCTGGAG 660 TGCTGGTGCT GGAGCAGTTC CTAGGTGCAC TGCCTGTGAA GCTCCGGACA TGGGTGGAA 720 CGCAGCACCC AGAGAACTGC CAAGAGGTGG TGGCCCTGGT AGAGGGTGTG ACCTGGATG 780 CTGAGGAGGA AGTACTTCCT GGCAGGACAA CCTGCCGAGG GCACCACCTG CTGCCTCGA 840 GTCACTGCCC AGCAGGAGGA GAAGCAGGAG GATGCAGCCA TCTGCCCAGT GACAGTGCT 900 CCTGAGGAGC CAGTGACCTT CCAGGATGTG GCTGTGGACT TCAGCCGGGA GGAGTGGGG 960 CTGCTGGGCC CGACACAGAG GACCGAGTAC CGCGATGTGA TGCTGGAGAC CTTTGGGC 1020 CTGGTCTCTG TGGGGTGGGA GACTACACTG GAAAATAAAG AGTTAGCTCC AAATTCTG 1080 ATTCCTGAGG AAGAACCAGC CCCCAGCCTG AAAGTACAAG AATCCTCAAG GGATTGTG 1140 TTGTCCTCTA CATTAGAAGA TACCTTGCAG GGTGGGGTCC AGGAAGTCCA AGACACAG 1200 TTGAAGCAGA TGGAGTCTGC TCAGGAAAAA GACCTTCCTC AGAAGAAGCA CTTTGACA 1260 CGTGAGTCCC AGGCAAACAG TGGTGCTCTT GACACAAACC AAGTTTCGCT CCAGAAAA 1320 GACAACCCTG AGTCCCAGGC AAACAGTGGC GCTCTTGACA CAAACCAAGT TTTGCTCC 1380 AAAATTCCTC CTAGAAAACG ATTGCGCAAA CGTGACTCAC AAGTTAAAAG TATGAAAC 1440 AATTCACGTG TAAAAATTCA TCAGAAGAGC TGTGAAAGGC AAAAGGCCAA GGAAGGCA 1500 GGTTGTAGGA AAACCTTCAG TCGGAGTACT AAACAGATTA CGTTTATAAG AATTCACA 1560 GGGAGCCAAG TTTGCCGATG CAGTGAATGT GGTAAAATAT TCCGGAACCC AAGATACT 1620 TCTGTGCATA AGAAAATCCA TACCGGAGAG AGGCCCTATG TGTGTCAAGA CTGTGGGA 1680 GGATTTGTTC AGAGCTCTTC CCTCACACAG CATCAGAGAG TTCATTCTGG AGAGAGAC 1740 TTTGAATGTC AGGAGTGTGG GAGGACCTTC AATGATCGCT CAGCCATCTC CCAGCACC 1800 AGGACTCACA CTGGCGCTAA GCCCTACAAG TGTCAGGACT GTGGAAAAGC CTTCCGCC 1860 AGCTCCCACC TCATCAGACA TCAGAGGACT CACACCGGGG AGCGCCCATA TGCATGCA 1920 AAATGTGGAA AGGCCTTCAC CCAGAGCTCA CACCTTATTG GGCACCAGAG AACCCACA 1980 AGGACAAAGC GAAAGAAGAA ACAGCCTACC TCATAGCTCT CAAGCCAGTT G 2031 1750 base pairs nucleic acid single linear HMC1NOT01 9476 54 GCCGCATGGT AACTCAGGCG CCGGGCGCAC TGTCCTAGCT GCTGGTTTTC CACGCTGGTT 60 TTAGCTCCCG GCGTCTGCAA AATGAAGATT GAGGAGGTGA AGAGCACTAC GAAGACGCA 120 CGCATCGCCT CCCACAGCCA CGTGAAAGGG CTGGGGCTGG ACGAGAGCGG CTTGGCCAA 180 CAGGCGGCCT CAGGGCTTGT GGGCCAGGAG AACGCGCGAG AGGCATGTGG CGTCATAGT 240 GAATTAATCG AAAGCAAGAA AATGGCTGGA AGAGCTGTCT TGTTGGCAGG ACCTCCTGG 300 ACTGGCAAGA CAGCTCTGGC TCTGGCTATT GCTCAGGAGC TGGGTAGTAA GGTCCCCTT 360 TGCCCAATGG TGGGGAGTGA AGTTTACTCA ACTGAGATCA AGAAGACAGA GGTGCTGAT 420 GAGAACTTCC GCAGGGCCAT TGGGCTGCGA ATAAAGGAGA CCAAGGAAGT TTATGAAGG 480 GAAGTCACAG AGCTAACTCC GTGTGAGACA GAGAATCCCA TGGGAGGATA TGGCAAAAC 540 ATTAGCCATG TGATCATAGG ACTCAAAACA GCCAAAGGAA CCAAACAGTT GAAACTGGA 600 CCCAGCATTT TTGAAAGTTT GCAGAAAGAG CGAGTAGAAG CTGGAGATGT GATTTACAT 660 GAAGCCAACA GTGGGGCCGT GAAGAGGCAG GGCAGGTGTG ATACCTATGC CACAGAATT 720 GACCTTGAAG CTGAAGAGTA TGTCCCCTTG CCAAAAGGGG ATGTGCACAA AAAGAAAGA 780 ATCATCCAAG ATGTGACCTT GCATGACTTG GATGTGGCTA ATGCGCGGCC CCAGGGGGG 840 CAAGATATCC TGTCCATGAT GGGCCAGCTA ATGAAGCCAA AGAAGACAGA AATCACAGA 900 AAACTTCGAG GGGAGATTAA TAAGGTGGTG AACAAGTACA TCGACCAGGG CATTGCTGA 960 CTGGTCCCGG GTGTGCTGTT TGTTGATGAG GTCCACATGC TGGACATTGA GTGCTTCA 1020 TACCTGCACC GCGCCCTGGA GTCTTCTATC GCTCCCATCG TCATCTTTGC ATCCAACC 1080 GGCAACTGTG TCATCAGAGG CACTGAGGAC ATCACATCCC CTCACGGCAT CCCTCTTG 1140 CTTCTGGACC GAGTGATGAT AATCCGGACC ATGCTGTATA CTCCACAGGA AATGAAAC 1200 ATCATTAAAA TCCGTGCCCA GACGGAAGGA ATCAACATCA GTGAGGAGGC ACTGAACC 1260 CTGGGGGAGA TTGGCACCAA GACCACACTG AGGTACTCAG TGCAGCTGCT GACCCCGG 1320 AACTTGCTTG CTAAAATCAA CGGGAAGGAC AGCATTGAGA AAGAGCATGT CGAAGAGA 1380 AGTGAACTTT TCTATGATGC CAAGTCCTCC GCCAAAATCC TGGCTGACCA GCAGGATA 1440 TACATGAAGT GAGATGGCTG AGGTTTTCAG CAGTAAGAGA CTCCCCAGGT GTGCCTGG 1500 TGGGTCCAGC CTGTGGGCGC TTGCCCCTGG GCTTGGGGCT GCCGTCCCCA CTCAGGCG 1560 GTCTGCAGCG CTGTCAGTTC AGTGTGGAAA GCATTTCTTT TTAAGTTATC GTAACTGT 1620 CTGTGGTTGC TTTGAAAGAA CCCTTCCTTA CCTGGTGTGT TTTCTATAAA TCTTCATA 1680 TTATTTTGAT TCTCTCTCTC TCTCTCTCTA AGTTTTTTAA AAATAAACTT TTCAGAAC 1740 AAAAAAAAAA 1750 1234 base pairs nucleic acid single linear THP1PLB01 10370 55 GGGCGGGGCC GGTCGGTGAG TCAGCGGCTC TCTGATCCAG CCCGGGAGAG GACCGAGCTG 60 GAGGAGCTGG GTGTGGGGTG CGTTGGGCTG GTGGGGAGGC CTAGTTTGGG TGCAAGTAG 120 TCTGATTGAG CTTGTGTTGT GCTGAAGGGA CAGCCCTGGG TCTAGGGGAG AGAGTCCCT 180 AGTGTGAGAC CCGCCTTCCC CGGTCCCAGC CCCTCCCAGT TCCCCCAGGG ACGGCCACT 240 CCTGGTCCCC GACGCAACCA TGGCTGAAGA ACAACCGCAG GTCGAATTGT TCGTGAAGG 300 TGGCAGTGAT GGGGCCAAGA TTGGGAACTG CCCATTCTCC CAGAGACTGT TCATGGTAC 360 GTGGCTCAAG GGAGTCACCT TCAATGTTAC CACCGTTGAC ACCAAAAGGC GGACCGAGA 420 AGTGCAGAAG CTGTGCCCAG GGGGGCAGCT CCCATTCCTG CTGTATGGCA CTGAAGTGC 480 CACAGACACC AACAAGATTG AGGAATTTCT GGAGGCAGTG CTGTGCCCTC CCAGGTACC 540 CAAGCTGGCA GCTCTGAACC CTGAGTCCAA CACAGCTGGG CTGGACATAT TTGCCAAAT 600 TTCTGCCTAC ATCAAGAATT CAAACCCAGC ACTCAATGAC AATCTGGAGA AGGGACTCC 660 GAAAGCCCTG AAGGTTTTAG ACAATTACTT AACATCCCCC CTCCCAGAAG AAGTGGATG 720 AACCAGTGCT GAAGATGAAG GTGTCTCTCA GAGGAAGTTT TTGGATGGCA ACGAGCTCA 780 CCTGGCTGAC TGCAACCTGT TGCCAAAGTT ACACATAGTA CAGGTGGTGT GTAAGAAGT 840 CCGGGGATTC ACCATCCCCG AGGCCTTCCG GGGAGTGCAT CGGTACTTGA GCAATGCCT 900 CGCCCGGGAA GAATTCGCTT CCACCTGTCC AGATGATGAG GAGATCGAGC TCGCCTATG 960 GCAAGTGGCA AAGGCCCTCA AATAAGCCCC TCCTGGGACT CCCTCAACCC CCTCCATT 1020 CTCCACAAAG GCCCTGGTGG TTTCCACATT GCTACCCAAT GGACACACTC CAAAATGG 1080 AGTGGGCAGG GAATCCTGGA GCACTTGTTC CGGGATGGTG TGGTGGAAGA GGGGATGA 1140 GAAAGAAATG GGGGGCCTGG GTCAGATTTT TATTGTGGGG TGGGATGAGT AGGACAAC 1200 ATTTCAGTAA TAAAATACAG AATAAAAAAA AAAA 1234 872 base pairs nucleic acid single linear THP1NOB01 30137 56 CACGCGTGCT GTCGGGGGAG GGATGCTGGG ACAGCTGCTC CCGCACACGG CTCGCGGTCT 60 CGGCGCCGCG GAGATGCCCG GCCAGGGTCC GGGGTCCGAC TGGACGGAGC GTAGCTCTT 120 TGCAGAGCCG CCCGCTGTGG CCGGGACCGA GGGTGGCGGC GGCGGATCAG CTGGATACT 180 TTGTTACCAG AATTCCAAAG GTTCTGATAG AATCAAAGAT GGATACAAAG TGAACTCAC 240 CATAGCTAAG CTGCAAGAGT TATGGAAAAC TCCCCAAAAT CAAACAATCC ACCTCTCTA 300 ATCAATGATG GAGGCGTCCT TTTTCAAGCA TCCAGACCTC ACCACAGGCC AGAAGCGTT 360 CCTGTGCAGC ATTGCTAAAA TCTATAATGC AAACTATCTG AAGATGTTAA TGAAGAGGC 420 GTACATGCAC GTACTTCAGC ACAGCTCACA AAAGCCAGGT GTCCTCACTC ATCACAGAA 480 CCGCCTTAGC TCCCGTTACT CACAGAAACA GCATTACCCT TGCACTACAT GGCGACATC 540 ACTGGAGAGA GAGGACTCGG GGTCTTCTGA TATCGCAGCT GCATCTGCAC CTGAAATGC 600 CATACAGCAT TCCCTTTGGC GGCCAGTGAG AAACAAAGAA GGGATAAAAA CTGGATATG 660 ATCTAAAACA AGATGTAAGT CACTGAAGAT TTTTAGAAGA CCAAGGAAAC TGTTCATGC 720 AACAGTTTCT TCAGATGATT CTGAATCACA CATGAGTGGA GAAAAAAAGG GAAGAGGAT 780 TACTACATAA TTTTATGCAA TCCATGTCAA TTGAGGACAA GGGGGACATC TGATGTTNA 840 TTGACAGTCT TGTCTCGTGT ATTGAATTCG TG 872 691 base pairs nucleic acid single linear SYNORAB01 77180 57 GGGAAAATGG CGCTGGCCAT GCTGGTCTTG GTGGTTTCGC CGTGGTCTGC GGCCCGGGGA 60 GTGCTTCGAA ACTACTGGGA GCGACTGCTA CGGAAGCTTC CGCAGAGCCG GCCGGGCTT 120 CCCAGTCCTC CGTGGGGACC AGCATTAGCA GTACAGGGCC CAGCCATGTT TACAGAGCC 180 GCAAATGATA CCAGTGGAAG TAAAGAGAAT TCCAGCCTTT TGGACAGTAT CTTTTGGAT 240 GCAGCTCCCA AAAATAGACG CACCATTGAA GTTAACCGGT GTAGGAGAAG AAATCCGCA 300 AAGCTTATTA AAGTTAAGAA CAACATAGAC GTTTGTCCTG AATGTGGTCA CCTGAAACA 360 AAACATGTCC TTTGTGCCTA CTGCTATGAA AAGGTGTGCA AGGAGACTGC AGAAATCAG 420 CGACAGATAG GGAAGCAAGA AGGGGGCCCT TTTAAGGCTC CCACCATAGA GACTGTGGT 480 CTGTACACAG GAGAGACACC GTCTGAACAA GATCAGGGCA AGAGGATCAT TGAACGAGA 540 AGAAAGCGAC CATCCTGGTT CACCCAGAAT TGACACCAAA GATGTTAAAA GGATAACTT 600 ACAGTAAATC ATTTCTCCTG AAATAGAGGA AGATTCTTTA CGTTGTTGTG CTTGTTTTT 660 AATCATCAGT ATAGTTTAAC ACATTCTTTC T 691 1994 base pairs nucleic acid single linear PITUNOR01 98974 58 CGGCTCGAGG CGTCTTGGCG GCAGTTGGTG GAACCGGAGC TTCGAGTCCG TCCCCGGTGC 60 TGCCTGCGCG TTCACCTGAG TCTCGCTGGA GCTCTTCTCG CCCGCCCACC TCATCTCAA 120 CCACTTTCCG CGGGGAGCGG CGCCAAGCTG GGCCTTCCTC GGATCAGGCG TCCCCTGAA 180 TCGGCACGCC CCTCTGCGTC CCCCTTCGGT CCCGCTAGGA CCCCGTCCGG GCTGCCGTC 240 CCTCGTCGCT ATGGCGCCCA CCATCCAGAC CCAGGCCCAG CGGGAGGATG GCCACAGGC 300 CAATTCCCAC CGGACTCTGC CTGAGAGGTC TGGAGTGGTC TGCCGAGTCA AGTACTGCA 360 TAGCCTCCCT GATATCCCCT TCGACCCCAA GTTCATCACC TACCCCTTCG ACCAGAACA 420 GTTCGTCCAG TACAAAGCCA CTTCCTTGGA GAAACAGCAC AAACATGACC TCCTGACTG 480 GCCAGACCTG GGGGTCACCA TCGATCTCAT CAATCCTGAC ACCTACCGCA TCGACCCCA 540 TGTTCTTCTA GATCCAGCTG ATGAGAAACT TTTGGAAGAG GAGATTCAGG CCCCCACCA 600 CTCCAAGAGA TCCCAGCAGC ATGCGAAGGT GGTGCCATGG ATGCGAAAGA CAGAGTACA 660 CTCCACTGAG TTCAACCGTT ATGGCATCTC CAATGAGAAG CCTGAGGTCA AGATTGGGG 720 TTCTGTGAAG CAGCAGTTTA CCGAGGAAGA AATATACAAA GACAGGGATA GCCAGATCA 780 AGCCATTGAG AAGACTTTTG AGGATGCCCA GAAATCAATC TCACAGCATT ACAGCAAAC 840 CCGAGTCACA CCGGTGGAGG TCATGCCTGT CTTCCCAGAC TTTAAGATGT GGATCAATC 900 ATGTGCTCAG GTGATCTTTG ACTCAGACCC AGCCCCCAAG GACACGAGTG GTGCAGCTG 960 GTTGGAGATG ATGTCTCAGG CCATGATTAG GGGCATGATG GATGAGGAAG GGAACCAG 1020 TGTGGCCTAT TTCCTGCCTG TAGAAGAGAC GTTGAAGAAA CGAAAGCGGG ACCAGGAG 1080 GGAGATGGAC TATGCACCAG ATGATGTGTA TGACTACAAA ATTGCTCGGG AGTACAAC 1140 GAACGTGAAG AACAAAGCTA GCAAGGGCTA TGAGGAAAAC TACTTCTTCA TCTTCCGA 1200 GGGTGACGGG GTTTACTACA ATGAGTTGGA AACCAGGGTC CGCCTTAGTA AGCGCCGG 1260 CAAGGCTGGG GTTCAGTCAG GCACCAACGC CCTGCTTGTG GTCAAACATC GGGACATG 1320 TGAGAAGGAA CTGGAAGCTC AGGAGGCACG GAAGGCCCAG CTAGAAAACC ACGAACCG 1380 GGAGGAAGAG GAAGAGGAGA TGGAGACAGA AGAGAAAGAA GCTGGGGGCT CAGATGAG 1440 GCAGGAGAAG GGCAGCAGCA GTGAGAAGGA GGGCAGTGAA GATGAGCACT CGGGCAGC 1500 GAGTGAACGG GAGGAAGGTG ACAGGGACGA GGCCAGTGAC AAGAGTGGCA GTGGTGAG 1560 CGAGAGCAGC GAGGATGAGG CCCGGGCTGC CCGTGACAAA GAGGAGATCT TTGGCAGT 1620 TGCTGATTCT GAGGACGATG CCGACTCTGA TGATGAGGAC AGAGGACAGG CCCAAGGT 1680 CAGTGACAAT GATTCAGACA GCGGCAGCAA TGGGGGTGGC CAGCGGAGCC GGAGCCAC 1740 CCGCAGCGCC AGTCCCTTCC CCAGTGGCAG CGAGCACTCG GCCCAGGAGG ATGGCAGT 1800 AGCTGCAGCT TCTGATTCCA GTGAAGCTGA TAGTGACAGT GACTGAGTCC CAGGGCAT 1860 AGGGCTGGTT CAGACACCAT TATTGTGAGC AGCAAAGCAC TTTTCTAGTG GTCTGTTT 1920 GAGCCTTTCA CTTGTTTGTT CCCCACCCCC AAACCTTTGC TGTTAATAAA GTCAACTT 1980 CTTTAAAAAA AAAA 1994 1594 base pairs nucleic acid single linear MUSCNOT01 118160 59 CCGCCCCCGC CGGGCGTGTG TGTCGTGTGT GTTTGGGGCC CGCGCGGGTT GCGCGCCCTC 60 CGCCTTCGCG CCTCCTGCCC CCGAGGCCCT ACTGCTGCCC CTGTGCCCCT CGCCCCGCC 120 GGCGTCGCGG GCCAACATGG GCCAGGAAGA GGAGCTGCTG AGGATCGCCA AAAAGCTGG 180 GAAGATGGTG GCCAGGAAGA ACACGGAAGG GGCCCTGGAC CTTCTGAAGA AGCTGCACA 240 CTGCCAGATG TCCATCCAGC TACTACAGAC AACCAGGATT GGAGTTGCTG TTAATGGGG 300 CCGCAAGCAC TGCTCAGACA AGGAGGTGGT GTCCTTGGCC AAAGTCCTTA TCAAAAACT 360 GAAGCGGCTG CTAGACTCCC CTGGACCCCC AAAAGGAGAA AAAGGAGAGG AAAGAGAAA 420 GGCAAAGAAG AAGGAAAAAG GGCTTGAGTG TTCAGACTGG AAGCCAGAAG CAGGCCTTT 480 TCCACCAAGG AAAAAACGAG AAGACCCCAA AACCAGGAGA GACTCTGTGG ACTCCAAGT 540 TTCTGCCTCC TCCTCTCCAA AAAGACCATC GGTGGAAAGA TCAAACAGCA GCAAATCAA 600 AGCGGAGAGC CCCAAAACAC CTAGCAGCCC CTTGACCCCC ACGTTTGCCT CTTCCATGT 660 TCTCCTGGCC CCCTGCTATC TCACAGGGGA CTCTGTCCGG GACAAGTGTG TGGAGATGC 720 GTCAGCAGCC CTGAAGGCGG ACGATGATTA CAAGGACTAT GGAGTCAACT GTGACAAGA 780 GGCATCAGAA ATCGAAGATC ATATCTACCA AGAGCTCAAG AGCACGGACA TGAAGTACC 840 GAACCGCGTG CGCAGCCGCA TAAGCAACCT CAAGGACCCC AGGAACCCCG GCCTGCGGC 900 GAACGTGCTC AGTGGGGCCA TCTCCGCAGG GCTTATAGCC AAGATGACGG CAGAGGAAA 960 GGCCAGTGAT GAACTGAGGG AGTTGAGGAA TGCCATGACC CAGGAGGCCA TCCGTGAG 1020 CCAGATGGCC AAGACTGGCG GCACCACCAC TGACCTCTTC CAGTGCAGCA AATGCAAG 1080 GAAGAACTGC ACCTATAACC AGGTGCAGAC ACGCAGTGCT GATGAGCCCA TGACTACC 1140 TGTCTTATGC AATGAATGTG GCAATCGCTG GAAGTTCTGC TGATGGAACA GCCAGCCA 1200 AACAAGGTGA GGAAGAAGAA AGAGGAAGCG CTGAATTATC TGAACTGGAG AAGCAATA 1260 AATTAAAGTG AAGGAAAATA CTGAACTCTG TCTGAGTGGG ATGGTATGAG TTAGAGGA 1320 AATTCTCTTG CAAATTAATA ATCGGTCATT AGAAACAATT GGTTAATGGG GGAGCCTA 1380 TGGAGAATGA TGCTGAGAAT TTGTATTGAT GAACCTCTTT TAGAAACTGC AGAGGGCT 1440 GCACGGTGGC TTATGGCTGT AATCTGCAAA CTCTGGGAGG CTGAGGTGGG AGAATCGC 1500 AACCCCAGAA GTTTGAGTCC AGCCCAGGCA ACACAGCAAG ACCCCATCTC TATAAAAA 1560 AAAAATAAAG AAATTGTAGA CGCCTCGGGG ACAT 1594 1460 base pairs nucleic acid single linear TLYMNOR01 140516 60 GGCCGTCCGG CCTCCCTGAC ATGCAGATTT CCACCCAGAA GACAGAGAAG GAGCCAGTGG 60 TCATGGAATG GGCTGGGGTC AAAGACTGGG TGCCTGGGAG CTGAGGTAGC CACCGTTTC 120 GCCTGGCCAG CCCTCTGGAC CCCGAGGTTG GACCCTACTG TGACACACCT ACCATGCGG 180 CACTCTTCAA CCTCCTCTGG CTTGCCCTGG CCTGCAGCCC TGTTCACACT ACCCTGTCA 240 AGTCAGATGC CAAAAAAGCC GCCTCAAAGA CGCTGCTGGA GAAGAGTCAG TTTTCAGAT 300 AGCCGGTGCA AGACCGGGGT TTGGTGGTGA CGGACCTCAA AGCTGAGAGT GTGGTTCTT 360 AGCATCGCAG CTACTGCTCG GCAAAGGCCC GGGACAGACA CTTTGCTGGG GATGTACTG 420 GCTATGTCAC TCCATGGAAC AGCCATGGCT ACGATGTCAC CAAGGTCTTT GGGAGCAAG 480 TCACACAGAT CTCACCCGTC TGGCTGCAGC TGAAGAGACG TGGCCGTGAG ATGTTTGAG 540 TCACGGGCCT CCACGACGTG GACCAAGGGT GGATGCGAGC TGTCAGGAAG CATGCCAAG 600 GCCTGCACAT AGTGCCTCGG CTCCTGTTTG AGGACTGGAC TTACGATGAT TTCCGGAAC 660 TCTTAGACAG TGAGGATGAG ATAGAGGAGC TGAGCAAGAC CGTGGTCCAG GTGGCAAAG 720 ACCAGCATTT CGATGGCTTC GTGGTGGAGG TCTGGAACCA GCTGCTAAGC CAGAAGCGC 780 TGGGCCTCAT CCACATGCTC ACCCACTTGG CCGAGGCTCT GCACCAGGCC CGGCTGCTG 840 CCCTCCTGGT CATCCCGCCT GCCATCACCC CCGGGACCGA CCAGCTGGGC ATGTTCACG 900 ACAAGGAGTT TGAGCAGCTG GCCCCCGTGC TGGATGGTTT CAGCCTCATG ACCTACGAC 960 ACTCTACAGC GCATCAGCCT GGCCCTAATG CACCCCTGTC CTGGGTTCGA GCCTGCGT 1020 AGGTCCTGGA CCCGAAGTCC AAGTGGCGAA GCAAAATCCT CCTGGGGCTC AACTTCTA 1080 GTATGGACTA CGCGACCTCC AAGGATGCCC GTGAGCCTGT TGTCGGGGCC AGGTACAT 1140 AGACACTGAA GGACCACAGG CCCCGGATGG TGTGGGACAG CCAGGCCTCA GAGCACTT 1200 TCGAGTACAA GAAGAGCCGC AGTGGGAGGC ACGTCGTCTT CTACCCAACC CTGAAGTC 1260 TGCAGGTGCG GCTGGAGCTG GCCCGGGAGC TGGGCGTTGG GGTCTCTATC TGGGAGCT 1320 GCCAGGGCCT GGACTACTTC TACGACCTGC TCTAGGTGGG CATTGCGGCC TCCGCGGT 1380 ACGTGTTCTT TTCTAAGCCA TGGAGTGAGT GAGCAGGTGT GAAATACAGG CCTCCACT 1440 GTTTGCTGTG AAAAAAAAAA 1460 1594 base pairs nucleic acid single linear SPLNNOT02 207452 61 CGGCTCGAGC GGCTCGAGCG GCTCGAGGCG GAGAGCGCGG AGCCGGGCCG CACCCGCCGA 60 GCCGTGAAAA AAGTACATCT CCTGGAAGGG ATGCTTTTTA GCTGAGCTCT GGTGGATGA 120 AGGAGCTAGC CTTGAAAAAC TTGATACTGA TGGACATTGT GTGGGCCAGA GGCAGGGAT 180 GTTGGCTATG ACCCCAAACC AGATGGCAGG AATAACACCA AGTTCCAGGT GGCAGTGGC 240 GGGTCTGTGT CTGGACTTGT TACTCGGGCG CTGATCAGTC CCTTCGACGT CATCAAGAT 300 CGTTTCCAGC TTCAGCATGA GCGCCTGTCT CGCAGTGACC CCAGCGCAAA GTACCATGG 360 ATCCTCCAGG CCTCTAGGCA GATTCTGCAG GAGGAGGGTC CGACAGCTTT CTGGAAAGG 420 CACGTCCCAG CTCAGATTCT CTCCATAGGC TATGGAGCTG TCCAATTCTT GTCATTTGA 480 ATGCTGACGG AGCTGGTCCA CAGAGGCAGC GTGTACGACG CCCGGGAATT CTCAGTGCA 540 TTTGTATGTG GTGGCCTGGC TGCCTGTATG GCCACCCTCA CTGTGCACCC CGTGGATGT 600 CTGCGCACCC GCTTTGCAGC TCAGGGTGAG CCCAAGGTCT ATAATACGCT GCGCCACGC 660 GTGGGGACCA TGTATAGGAG CGAAGGCCCC CAGGTTTTCT ACAAAGGCTT GGCTCCCAC 720 TTGATCGCCA TCTTCCCCTA CGCCGGGCTG CAGTTCTCTT GCTACAGCTC CTTGAAGCA 780 CTGTACAAGT GGGCCATACC AGCCGAAGGA AAGAAAAATG AGAACCTCCA AAACCTGCT 840 TGTGGCAGTG GAGCTGGTGT CATCAGCAAG ACCCTGACAT ATCCGCTGGA CCTCTTCAA 900 AAGCGGCTAC AGGTTGGAGG GTTTGAGCAT GCCAGAGCTG CCTTTGGCCA GGTACGGAG 960 TACAAGGGCC TCATGGACTG TGCCAAGCAG GTGCTGCAAA AGGAAGGCGC CCTGGGCT 1020 TTCAAGGGCC TGTCCCCCAG CTTGCTGAAG GCTGCCCTCT CCACAGGCTT CATGTTCT 1080 TCGTATGAAT TCTTCTGTAA TGTCTTCCAC TGCATGAACA GGACAGCCAG CCAGCGCT 1140 GCGCAGGAAG GACCCCAGGT CTTCCCTGGA GGCAGCCTCC TGAAGGAAGG AAGATTCA 1200 CTCCACTGAG AGGTGCCGTC TGGCCCTTCC CTGCAGGCCA GCTGCCCCAA GCGGGGTA 1260 AGCCTTGAAC CCACCCAGCT GGGACACCAC CAGAAGGTCC AGGGCTCTCC CCATGAGA 1320 ATCAGAGGGA TGCAGGACGT GGTCTATGGT GAGCCAACGA CACAGTGAGA AGGAGCAG 1380 AGTTGCTGTT TCTCCTCTGA CCAGCCCACA CTGCAAAGGA AACAGACGCC ATCCTACA 1440 TATCAGCCCT GCCTGCCAGG AGAACAGAGC ACACTCCTGG TCTGGATGGG GCTGCTGC 1500 GAGTGCAGAG GGCTGCGGTA GGCCCTTTGC AGGAGTCAGG TCCCTACACT TGGCCTGT 1560 GTGCAACCTA TTTAATAGAC GATTAAAGCC TAGA 1594 1249 base pairs nucleic acid single linear SPLNNOT02 208836 62 TCTGCTAGGG CACAAGAGAG ACGGGCGCTC GCGCTCTCGC AGTCCTCTTC CGTCAGTGTC 60 TTTTGCTTCG ACTCCCGGCG GAGCGCGCAA CGTGGAGTGA CGTGCAGGGG CCAAGTGCA 120 CCCAGGCAGC CACGGCTGTT TCGGAGCTCA GGACTCTAAA ATGGCAGAGC AGCTTTCTC 180 AGGAAAGGCG GTGGATCAGG TGTGCACCTT CCTTTTCAAA AAGCCTGGGC GGAAAGGGG 240 TGCTGGACGC AGAAAGCGCC CGGCCTGCGA CCCAGAGCCC GGAGAAAGCG GCAGCAGTA 300 CGACGAAGGC TGCACTGTGG TTCGACCGGA AAAGAAGCGG GTGACCCACA ATCCAATGA 360 GCAGAAGACC CGTGACAGTG GTAAACAGAA GGCGGCTTAC GGCGACTTGA GCAGCGAAG 420 GGAAGAGGAA AATGAGCCCG AGAGTCTCGG CGTGGTTTAT AAATCCACCC GTTCGGCGA 480 ACCCGTGGGA CCAGAGGATA TGGGAGCGAC AGCCGTCTAT GAGCTGGACA CAGAGAAAG 540 GCGCGATGCA CAAGCCATCT TTGAGCGCAG CCAGAAGATC CAGGAGGAGC TGAGGGGCA 600 GGAGGATGAC AAGATCTATC GGGGAATCAA CAATTATCAG AAATACATGA AGCCCAAGG 660 TACGTCTATG GGCAATGCCT CTTCCGGGAT GGTGAGGAAG GGCCCCATCC GAGCGCCCG 720 GCATCTACGT GCCACCGTGC GCTGGGATTA CCAGCCCGAC ATCTGTAAGG ACTACAAAG 780 GACTGGCTTC TGCGGCTTCG GAGACAGCTG CAAATTCCTC CATGACCGTT CAGATTACA 840 GCATGGGTGG CAGATCGAAC GTGAGCTTGA TGAGGGTCGC TATGGTGTCT ATGAGGATG 900 AAACTATGAA GTGGGAAGCG ATGATGAGGA AATACCATTC AAGTGTTTCA TCTGTCGCC 960 GAGCTTCCAA AACCCAGTTG TCACCAAGTG CAGGCATTAT TTCTGCGAGA GCTGTGCA 1020 GCAGCATTTC CGCACCACCC CGCGCTGCTA TGTCTGTGAC CAGCAGACCA ATGGCGTC 1080 CAATCCAGCG AAAGAATTGA TTGCTAAACT AGAGAAGCAT CGAGCTACAG GAGAGGGT 1140 TGCTTCCGAC TTGCCAGAAG ACCCCGATGA GGATGCAATT CCCATTACTT AGGTTTCC 1200 TAATTCTTAA ATTTAAAAAA TAAACGTTTT GTTCTTTTGG AAAAAAAAA 1249 1309 base pairs nucleic acid single linear MMLR3DT01 569710 63 CAGGCCCGCA GACCGGAAGC AGCCCGCGCC GGGGGCTTCT GGGAAAAGGC TTGTGAACGG 60 CGTTTCTGCG TCTGCCGTGG ACAGCGAANT GCTTGCGGTT CCTGAGCCGG AGGAGTGCC 120 GTGAAGAAAA CGGGGTATTG CCCTGAGGCT TATATTCTGC CTCAGTTGTC TTTTCTTGA 180 ATATTATAAA TCAGAATGTC TGCACAGTCA GTGGAAGAAG ATTCAATACT TATCATCCC 240 ACTCCAGATG AAGAGGAAAA AATTCTGAGA GTGAAGTTGG AGGAGGATCC TGATGGCGA 300 GAGGGATCAA GTATCCCCTG GAACCATCTC CCAGACCCAG AGATTTTCCG ACAGCGATT 360 AGGCAGTTTG GATACCAGGA TTCACCTGGG CCCCGTGAGG CTGTGAGCCA GCTCCGAGA 420 CTTTGCCGTC TGTGGCTCAG GCCAGAGACG CACACAAAAG AACAAATCTT GGAGCTGGT 480 GTGCTGGAGC AGTTTGTTGC CATCCTACCC AAAGAGCTAC AGACTTGGGT TCGAGATCA 540 CATCCAGAGA ATGGAGAGGA GGCAGTGACA GTGCTGGAGG ATTTGGAGAG TGAACTTGA 600 GACCCTGGAC AACCGGTTTC TCTCCGTCGA CGAAAACGGG AAGTACTAGT AGAAGACAT 660 GTATCTCAAG AAGAAGCTCA GGGATTACCA AGTTCTGAGC TTGATGCTGT GGAGAACCA 720 CTCAAGTGGG CATCCTGGGA GCTCCATTCC CTAAGGCACT GTGATGATGA TGGTAGGAC 780 GAAAATGGAG CACTAGCTCC AAAGCAGGAG CTTCCTTCAG CATTAGAATC CCATGAAGT 840 CCTGGCACTC TCAGTATGGG TGTTCCTCAA ATTTTTAAAT ATGGAGAAAC CTGTTTCCC 900 AAGGGCAGGT TTGAAAGAAA GAGAAATCCC TCTCGAAAGA AACAACATAT ATGTGATGA 960 TGTGGAAAAC ACTTCAGTCA GGGCTCAGCC CTTATTCTTC ATCAAAGAAT TCACAGTG 1020 GAGAAACCTT ATGGATGTGT TGAGTGTGGG AAAGCATTCA GCCGAAGTTC CATTCTTG 1080 CAACACCAGA GAGTCCACAC TGGAGAAAAA CCTTACAAAT GTCTTGAATG TGGGAAAG 1140 TTTAGCCAGA ATTCGGGGCT TATTAATCAT CAGAGAATCC ATACTGGGGA GAAACCTT 1200 GAATGCGTTC AGTGTGGGAA ATCGTATAGT CAAAGCTCAA ATCTTTTTAG ACATCAGA 1260 AGACACAATG CAGAAAAACT TCTGAATGTT GTGAAAGTTT AAGAAATTG 1309 76 base pairs nucleic acid single linear BRSTTUT01 606742 64 TTTTTTTTTT TTTTTTGTTG GAAAGGAGAT GTTTATTTTC TTCTTCCCAT GCTATGGAAG 60 GACATTGTAT TCCGCA 76 1327 base pairs nucleic acid single linear COLNNOT01 611135 65 CCTACCCTCT TCTGTTGCTT TCTCCCTGTG GCTCGCGCCG TCCCCCGCCG CCCGTCGACC 60 CCGCTTCCAT GTCCCTGGCG GACACAGCTC CCAGGAACCT CCACGCCCAT GGCCACTAG 120 CAGAGGGAAT CCTCTATCAC CTCCTGCTGT TCCACCTCGA GCTGCGACGC AGACGACGA 180 GGCGTGCGCG GCACCTGCGA AGATGCTTCC CTGTGCAAGA GGTTTGCAGT AAGCATTGG 240 TACTGGCATG ACCCTTACAT ACAGCACTTT GTGAGACTGT CTAAAGAGAG GAAAGCCCC 300 GAAATCAACA GAGGATATTT TGCTCGAGTC CATGGTGTCA GTCAGCTTAT AAAGGCATT 360 CTACGGAAGA CAGAATGTCA TTGTCAAATT GTCAACCTTG GGGCAGGCAT GGATACCAC 420 TTCTGGAGAT TAAAGGATGA AGATCTTCTC CCAAGTAAAT ATTTTGAGGT TGACTTTCC 480 ATGATTGTCA CGAGAAAGCT GCACAGTATC AAATGCAAGC CTCCCCTATC CAGCCCCAT 540 CTAGAACTGC ATTCAGAGGA CACACTTCAG ATGGATGGAC ACATACTGGA TTCAAAGAG 600 TATGCCGTTA TTGGAGCAGA TCTCCGAGAC CTGTCTGAAC TGGAAGAGAA GCTAAAGAA 660 TGTAACATGA ATACACAATT GCCAACACTC CTGATAGCTG AATGTGTGCT GGTTTACAT 720 ACTCCAGAGC AGTCCGCAAA CCTCCTGAAG TGGGCAGCCA ACAGTTTTGA GAGAGCCAT 780 TTCATAAACT ACGAACAGGT GAACATGGGT GATCGGTTTG GGCAGATCAT GATTGAAAA 840 CTGCGGAGAC GCCAGTGTGA CCTGGCGGGA GTGGAGACCT GCAAGTCATT AGAGTCACA 900 AAAGAACGGC TCCTGTCGAA TGGGTGGGAA ACAGCATCGG CCGTCGACAT GATGGAGTT 960 TACAACAGGT TACCTCGAGC TGAAGTGAGC AGGATAGAAT CACTTGAATT CCTGGATG 1020 ATGGAGCTGC TGGAGCAGCT CATGCGGCAT TACTGCCTTT GCTGGGCAAC CAAAGGAG 1080 AATGAGCTTG GGCTGAAGGA GATAACTTAT TAATCTGTCG AAGGCTTATG CCGAGCCA 1140 AGCCGAAGCC ACTTGCCCTC CTGGAGGAGA CCTGCAAGCT CCCTGAGCGG TGGGCGGG 1200 TCGTCCGCAG GTCTCATCCC ACACTCTTGA GAAGCCTTGG TCACTACAGT GGTCGCAC 1260 GTTCCTCTTC CTGTTCCTGT TGACATGTCG TTGTTTAAAT AAATCTCACT TGCCACCA 1320 AAAAAAA 1327 1892 base pairs nucleic acid single linear BRSTNOT03 641127 66 AGGCGTGGGG TTCCACGTGG GAGGTGAGAC TGGCCGGGCT TCCCCAGCCG CTGGCAGAGG 60 CGTTCAGGGG TCAGGAAATG GGTGTGCCGC GGGAGACTTG AATGAAAATT CTGTGGAGA 120 TCTTCAACAG AAAACACTTC AGGATCTGTT ACATGAGCTT TCCTCCTGGC TAGTTTTGG 180 AGGCATGGCC AGTACAATTA CTGGAAGTCA GGATTGTATT GTGAATCATC GAGGGGAAG 240 GGATGGGGAG CCTGAACTAG ATATTTCCCC TTGTCAACAG TGGGGAGAAG CATCTTCTC 300 TATTTCCAGA AACAGGGACA GTGTGATGAC TCTTCAAAGT GGTTGTTTCG AAAACATTG 360 AAGTGAAACA TATTTGCCTT TGAAAGTCTC AAGCCAAATA GACACACAAG ACTCTTCAG 420 GAAGTTCTGT AAGAATGAGC CTCAGGATCA TCAGGAAAGC AGACGTCTCT TTGTAATGG 480 AGAAAGCACT GAGAGAAAAG TGATAAAGGG GGAAAGTTGT TCAGAGAACC TTCAAGTTA 540 ACTGGTGTCT GATGGACAAG AACTGGCCTC GCCATTGTTA AATGGTGAGG CAACTTGCC 600 GAATGGCCAG TTAAAAGAAT CTTTGGATCC CATTGACTGT AACTGCAAAG ACATTCATG 660 ATGGAAATCA CAGGTGGTCA GTTGTAGTCA GCAGAGAGGT CATACAGAGG AGAAACCCT 720 TGACCATAAT AACTGTGGGA AAATACTTAA CACCAGCCCA GATGGTCATC CATATGAGA 780 AATCCACACT GCAGAGAAAC AATACGAAGG TAGTCAGTGT GGTAAGAACT TCAGTCAAA 840 CTCAGAGCTA CTACTTCATC AGAGAGACCA CACAGAAGAA AAACCCTACA AATGTGAGC 900 ATGTGGGAAG GGCTTCACAA GGAGCTCGAG TCTGCTTATC CATCAGGCAG TCCACACAG 960 TGAGAAGCCT TATAAGTGTG ACAAGTGTGG GAAGGGCTTC ACCAGGAGCT CAAGTCTG 1020 CATCCATCAT GCCGTCCATA CAGGCGAAAA ACCTTATAAA TGTGACAAGT GTGGGAAG 1080 CTTTAGTCAG AGCTCCAAAC TGCACATCCA CCAGCGAGTC CACACTGGAG AGAAGCCC 1140 TGAGTGTGAG GAGTGTGGTA TGAGCTTCAG TCAGCGCTCA AACCTGCACA TCCACCAG 1200 AGTACACACA GGAGAGAGGC CCTACAAGTG TGGTGAGTGT GGGAAGGGCT TCAGTCAG 1260 CTCGAACCTT CACATTCACC GGTGCATCCA CACAGGAGAG AAGCCTTACC AATGCTAT 1320 GTGTGGGAAG GGTTTCAGCC AGAGCTCGGA TCTTCGCATC CATCTCAGAG TCCACACT 1380 AGAGAAGCCC TATCACTGTG GCAAGTGTGG GAAGGGATTT AGCCAGAGTT CCAAACTC 1440 CATCCACCAG AGAGTACATA CTGGAGAGAA GCCCTATGAG TGCAGCAAGT GTGGGAAG 1500 CTTCAGCCAG AGCTCCAACC TTCACATCCA CCAGCGGGTT CACAAGAGAG ATCCTCGA 1560 CCATCCAGGT CTTCACAGCG CTCATACTGT AAACACTGTT AAATATTTAG TATCACTC 1620 ACTTTATATT CTACAAAGGA GAGAGATGTA AGGGTTATTT AGATATGTTC CCTCACTG 1680 AAATCACTCA TTCAACATAT TTAAGTATCA AGCACTTTGT TATGCTGTAC AATGAATG 1740 TTGCTCTTGT TTCTCAGATG GGTAGAGTAA AAGTGTCTGT ACTTTACCGT TCAACTAC 1800 GTTCTACCCA GCATTTTAAC GGCAAGAACT TTATATTTAT TCTCATAGCA GGGCATGT 1860 CCCTTTGATC ACAGGCTCTG AGAATGCTTT AT 1892 843 base pairs nucleic acid single linear LUNGTUT02 691768 67 GGAAGTGTCT TCAGGGAGAG GAAGCCGGCG GCCTCACTGC TATGGGCCGC AACAAGAAGA 60 AGAAGCGAGA TGGTGACGAC CGGCGGCCGA GGCTCGTTCT TAGCTTCGAC GAGGAGAAG 120 GGCGGGAGTA CCTGACAGGC TTCCACAAGC GGAAGGTCGA GCGAAAGAAG GCAGCCATT 180 AGGAGATTAA GCAGCGGCTG AAAGAGGAGC AGAGGAAGCT TCGGGAGGAG CGCCACCAG 240 AATACTTGAA GATGCTGGCA GAGAGAGAAG AGGCTCTGGA GGAGGCAGAT GAGCTGGAC 300 GGTTGGTGAC AGCAAAGACG GAGTCGGTGC AGTATGACCA CCCCAACCAC ACAGTCACC 360 TGACCACCAT CAGTGACCTG GACCTCTCGG GGGCCCGGCT GCTCGGGCTG ACCCCACCT 420 AGGGAGGGGC TGGAGACAGG TCTGAGGAGG AGGCGTCATC CACGGAGAAA CCAACCAAA 480 CCTTGCCCAG GAAGTCCAGA GACCCCCTGC TCTCTCAGCG GATCTCCTCC CTCACAGCA 540 CACTACATGC ACACAGCCGC AAAAAGGTCA AGAGGAAACA TTCCCGACGG GCCCAGGAC 600 CCAAAAAGCC CCCAAAGGGC CCTTCGTACC AGCAAAGGCC CAGCGGCGCC GTCTTCACA 660 GCAAAGCACC GGCACAGCGG GGGAATTNAA GANCCGAGAA CGAAGCCGGT TGCCCCCAT 720 CTAAGGCTTN CCGGGGANCT TGTTCCCTTG GTTCAGCTTT GGCTGTTCCC CTGTTAGNC 780 CAGCCTTGNA ACTTAAGGTG TTGCCTTAAC CGGCATTGTT TGCCCGCTTG GCTGGTTTT 840 TTG 843 1643 base pairs nucleic acid single linear SYNOOAT01 724157 68 GGGAGGCTGA AGCAAGAGAA CCGCTTGAAC GGGGGTGGAT GTTGCAGTGA GCCAAGATGG 60 TGCCACTGCA CTCCAGCCTG GCAACAGTGC GCAGAGCGAG ACGCCGTCTC AAAACAAAA 120 TCTCACAGTG GCCCAGCCTC CTTTCCTGCC ATCCCTGGAG TTGTGGTCTG TTTGAGGTT 180 GGTTTTCAGG ACTGAAGCTT CAAGATGGCT GACCAGGACC CTGCGGGCAT CAGCCCCCT 240 CAGCAAATGG TGGCCTCAGG CACCGGGGCT GTGGTTACCT CTCTCTTCAT GACACCCCT 300 GACGTGGTGA AGGTTCGCCT GCAGTCTCAG CGGCCCTCCA TGGCCAGCGA GCTGATGCC 360 TCCTCCAGAC TGTGGAGCCT CTCCTATACC AAATGGAAGT GCCTCCTGTA TTGCAATGG 420 GTCCTGGAGC CTCTGTACCT GTGCCCAAAT GGTGCCCGCT GTGCCACCTG GTTTCAAGA 480 CCTACCCGCT TCACTGGCAC CATGGATGCC TTCGTGAAGA TCGTGAGGCA CGAGGGCAC 540 AGGACCCTCT GGAGCGGCCT CCCCGCCACC CTGGTGATGA CTGTGCCAGC TACCGCCAT 600 TACTTCACTG CCTATGACCA ACTGAAGGCC TTCCTGTGTG GTCGAGCCCT GACCTCTGA 660 CTCTACGCAC CCATGGTGGC TGGCGCGCTG GCCCGCTTGG GCACCGTGAC TGTGATCAG 720 CCCCTGGAGC TTATGCGGAC AAAGCTGCAG GCTCAGCATG TGTCGTACCG GGAGCTGGG 780 GCCTGTGTTC GAACTGCAGT GGCTCAGGGT GGCTGGCGCT CACTGTGGCT GGGCTGGGG 840 CCCACTGCCC TTCGAGATGT GCCCTTCTCA GCCCTGTACT GGTTCAACTA TGAGCTGGT 900 AAGAGCTGGC TCAATGGGCT CAGGCCGAAG GACCAGACTT CTGTGGGCAT GAGCTTTGT 960 GCTGGTGGCA TCTCAGGGAC GGTGGCTGCA GTGCTGACTC TACCCTTTGA CGTGGTAA 1020 ACCCAACGCC AGGTCGCTCT GGGAGCGATG GAGGCTGTGA GAGTGAACCC CCTGCATG 1080 GACTCCACCT GGCTGCTGCT GCGGAGGATC CGGGCCGAGT CGGGCACCAA GGGACTCT 1140 GCAGGCTTCC TTCCTCGGAT CATCAAGGCT GCCCCCTCCT GTGCCATCAT GATCAGCA 1200 TATGAGTTCG GCAAAAGCTT CTTCCAGAGG CTGAACCAGG ACCGGCTTCT GGGCGGCT 1260 AAGGGGCAAG GAGGCAAGGA CCCCGTCTCT CCCACGGATG GGGAGAGGGC AGGAGGAG 1320 CCAGCCAAGT GCCTTTTCCT CAGCACTGAG GGAGGGGGCT TGTTTCCCTT CCCTCCCG 1380 GACAAGCTCC AGGGCAGGGC TGTCCCTCTG GGCGGCCCAG CACTTCCTCA GACACAAC 1440 CTTCCTGCTG CTCCAGTCGT GGGGATCATC ACTTACCCAC CCCCCAAGTT CAAGACCA 1500 TCTTCCAGCT GCCCCCTTCG TGTTTCCCTG TGTTTGCTGT AGCTGGGCAT GTCTCCAG 1560 ACCAAGAAGC CCTCAGCCTG GTGTAGTCTC CCTGACCCTT GTTAATTCCT TAAGTCTA 1620 GATGATGAAC TTCAAAAAAA AAA 1643 2029 base pairs nucleic acid single linear BRAITUT03 864683 69 GCAGCTGTGA GGGAGTCGCT GTGATCCGGG GCCCCGGAAC CCGAGCTGGA GCTGAAGCGC 60 AGGCTGCGGG GCGCGGAGTC GGGAGTGCAG GCCTGAGTGT TCCTTCCAGC ATGTCGGAG 120 GGGAGTCCCA GACAGTACTT AGCAGTGGCT CAGACCCAAA GGTAGAATCC TCATCTTCA 180 CTCCTGGCCT GACATCAGTG TCACCTCCTG TGACCTCCAC AACCTCAGCT GCTTCCCCA 240 AGGAAGAAGA AGAAAGTGAA GATGAGTCTG AGATTTTGGA AGAGTCGCCC TGTGGGCGC 300 GGCAGAAGAG GCGAGAAGAG GTGAATCAAC GGAATGTACC AGGTATTGAC AGTGCATAC 360 TGGCCATGGA TACAGAGGAA GGTGTAGAGG TTGTGTGGAA TGAGGTACAG TTCTCTGAA 420 GCAAGAACTA CAAGCTGCAG GAGGAAAAGG TTCGTGCTGT GTTTGATAAT CTGATTCAA 480 TGGAGCATCT TAACATTGTT AAGTTTCACA AATATTGGGC TGACATTAAA GAGAACAAG 540 CCAGGGTCAT TTTTATCACA GAATACATGT CATCTGGGAG TCTGAAGCAA TTTCTGAAG 600 AGACCAAAAA GAACCACAAG ACGATGAATG AAAAGGCATG GAAGCGTTGG TGCACACAA 660 TCCTCTCTGC CCTAAGCTAC CTGCACTCCT GTGACCCCCC CATCATCCAT GGGAACCTG 720 CCTGTGACAC CATCTTCATC CAGCACAACG GACTCATCAA GATTGGCTCT GTGGCTCCT 780 ACACTATCAA CAATCATGTG AAGACTTGTC GAGAAGAGCA GAAGAATCTA CACTTCTTT 840 CACCAGAGTA TGGAGAAGTC ACTAATGTGA CAACAGCAGT GGACATCTAC TCCTTTGGC 900 TGTGTGCACT GGAGATGGCA GTGCTGGAGA TTCAGGGCAA TGGAGAGTCC TCATATGTG 960 CACAGGAAGC CATCAGCAGT GCCATCCAGC TTCTAGAAGA CCCATTACAG AGGGAGTT 1020 TTCAAAAGTG CCTGCAGTCT GAGCCTGCTC GCAGACCAAC AGCCAGAGAA CTTCTGTT 1080 ACCCAGCATT GTTTGAAGTG CCCTCGCTCA AACTCCTTGC GGCCCACTGC ATTGTGGG 1140 ACCAACACAT GATCCCAGAG AACGCTCTAG AGGAGATCAC CAAAAACATG GATACTAG 1200 CCGTACTGGC TGAAATCCCT GCAGGACCAG GAAGAGAACC AGTTCAGACT TTGTACTC 1260 AGTCACCAGC TCTGGAATTA GATAAATTCC TTGAAGATGT CAGGAATGGG ATCTATCC 1320 TGACAGCCTT TGGGCTGCCT CGGCCCCAGC AGCCACAGCA GGAGGAGGTG ACATCACC 1380 TCGTGCCCCC CTCTGTCAAG ACTCCGACAC CTGAACCAGC TGAGGTGGAG ACTCGCAA 1440 TGGTGCTGAT GCAGTGCAAC ATTGAGTCGG TGGAGGAGGG AGTCAAACAC CACCTGAC 1500 TTCTGCTGAA GTTGGAGGAC AAACTGAACC GGCACCTGAG CTGTGACCTG ATGCCAAA 1560 AGAATATCCC CGAGTTGGCG GCTGAGCTGG TGCAGCTGGG CTTCATTAGT GAGGCTGA 1620 AGAGCCGGTT GACTTCTCTG CTAGAAGAGA CCTTGAACAA GTTCAATTTT GCCAGGAA 1680 GTACCCTCAA CTCAGCCGCT GTCACCGTCT CCTCTTAGAG CTCACTCGGG CCAGGCCC 1740 ATCTGCGCTG TGGCTGTCCC TGGACGTGCT GCAGCCCTCC TGTCCCTTCC CCCCAGTC 1800 TATTACCCTG TGAAGCCCCT TCCCTCCTTT ATTATTCAGG AGGGCTGGGG GGGCTCCC 1860 GTTCTGAGCA TCATCCTTTC CCCTCCCCTC TCTTCCTCCC CTCTGCACTT TGTTTACT 1920 TTTTGCACAG ACGTGGGCCT GGGCCTTCTC AGCAGCCGCC TTCTAGTTGG GGGCTAGT 1980 CTGATCTGCC GGCTCCCGCC CAGCCTGTGT GGAAAGGAGG CCCACGGGC 2029 821 base pairs nucleic acid single linear CERVNOT01 933353 70 CGGGTCGCCG CTGCGCCGGG CCGGGATGGC GGCCACCGCG CTGCTGGAGG CCGGCCTGGC 60 GCGGGTGCTC TTCTACCCGA CGCTGCTCTA CACCCTGTTC CGCGGGAAGG TGCCGGGTC 120 GGCGCACCGG GACTGGTACC ACCGCATCGA CCCCACCGTG CTGCTGGGCG CGCTGCCGT 180 GCGGAGCTTG ACGCGCCAGC TGGTACAGGA CGAGAACGTG CGCGGGGTGA TCACCATGA 240 CGAGGAGTAC GAGACGAGGT TCCTGTGCAA CTCTTCACAG GAGTGGAAGA GACTAGGAG 300 TGAGCAGCTG CGGCTCAGCA CAGTAGACAT GACTGGGATC CCCACCTTGG ACAACCTCC 360 GAAGGGAGTC CAATTTGCTC TCAAGTACCA GTCGCTGGGC CAGTGTGTTT ACGTGCATT 420 TAAGGCTGGG CGCTCCAGGA GTGCCACTAT GGTGGCAGCA TACCTGATTC AGGTGCACA 480 ATGGAGTCCA GAGGAGGCTG TAAGAGCCAT CGCCAAGATC CGGTCATACA TCCACATCA 540 GCCTGGCCAG CTGGATGTTC TTAAAGAGTT CCACAAGCAG ATTACTGCAC GGGCAACAA 600 GGATGGGACT TTTGTCATTT CAAAGACATG ATGTATGGGG ATTAGAAAGA ACTCAAGAC 660 CTCCTGCTTG ATACAGAACA AAAAGAGCTT AACAGGACCA ACAGGGCTTA AGCCCAGAC 720 TGACGTAACA GAAATGTGCC AATAGGTAAT AGGTAATTTT TCTTTCTCTG ACTTGTTTT 780 TTTTCTTGAA ATAACACTGT TGTGTGGCTA GAAAAAAAAA A 821 1139 base pairs nucleic acid single linear LATRTUT02 1404643 71 CGGCGACGGT GGGGAAGATG GCGTACCAGA GCTTGCGGCT GGAGTACCTG CAGATCCCAC 60 CGGTCAGCCG CGCCTACACC ACTGCCTGCG TCCTCACCAC CGCCGCCGTG CAGTTGGAA 120 TGATCACACC TTTTCAGTTG TACTTCAATC CTGAATTAAT CTTTAAACAC TTTCAAATA 180 GGAGATTAAT CACCAACTTC TTATTTTTTG GGCCAGTTGG ATTCAATTTT TTATTTAAC 240 TGATTTTTCT ATATCGTTAC TGTCGAATGC TAGAAGAAGG CTCTTTCCGA GGTCGGACA 300 CAGACTTTGT ATTTATGTTC CTTTTTGGTG GATTCTTAAT GACCCTTTTT GGTCTGTTT 360 TGAGCTTAGT TTTCTTGGGC CAGGCCTTTA CAATAATGCT CGTCTATGTG TGGAGCCGA 420 GGAACCCCTA TGTCCGCATG AACTTCTTCG GCCTTCTCAA CTTCCAGGCC CCCTTTCTG 480 CCTGGGTGCT CATGGGATTT TCCTTGTTGT TGGGGAACTC AATCATTGTG GACCTTTTG 540 GTATTGCAGT TGGACACATA TATTTTTTCT TGGAAGATGT ATTTCCCAAT CAACCTGGT 600 GAATAAGAAT TCTGAAAACA CCATCTATTT TGAAAGCTAT TTTTGATACA CCAGATGAG 660 ATCCAAATTA CAATCCACTA CCTGAGGAAC GGCCAGGAGG CTTCGCCTGG GGTGAGGGC 720 AGCGGCTTGG AGGTTAAAGC AGCAGTGCCA ATAATGAGAC CCAGCTGGGA AGGACTCGG 780 GATACCCACT GGGATCTTTT ATCCTTTGTT GCAAAAGTGT GGACACTTTT GACAGCTTG 840 CAGATTTTAA CTCCAGAAGC ACTTTATGAA ATGGTACACT GACTAATCCA GAAGACATT 900 CCAACAGTTT GCCAGTGGTT CCTCACTACA CTGGTACTGA AAGTGTAATT TCTTAGAGC 960 AAAAAACTGG AGAAACAAAT ATCCTGCCAC CTCTAACAAG TACATGAGTA CTTGATTT 1020 ATGGTATAAG GCAGAGCCTT TTCTTCCTCT TCTTGATAGA TGAGGCCATG GTGTAAAT 1080 AAGTTTCAGA GAGGACAAAA TAAAACGGAA TTCCATTTTT CTCTCACTGT AAAAAAAA 1139 1406 base pairs nucleic acid single linear SPLNNOT04 1561587 72 GGGCTGCCCC CGGGGGGCTG GCGGACTGGG CCGCGGGGGC CCCGGGGCCG GCGGTGCCGG60 GGTCATCGGG ATGATGCGGA CGCAGTGTCT GCTGGGGCTG CGCACGTTCG TGGCCTTCG120 CGCCAAGCTC TGGAGCTTCT TCATTTACCT TTTGCGGAGG CAGATCCGCA CGGTAATTC180 GTACCAAACT GTTCGATATG ATATCCTCCC CTTATCTCCT GTGTCCCGGA ATCGGCTAG240 CCAGGTGAAG AGGAAGATCC TGGTGCTGGA TCTGGATGAG ACACTTATTC ACTCCCACC300 TGATGGGGTC CTGAGGCCCA CAGTCCGGCC TGGTACGCCT CCTGACTTCA TCCTCAAGG360 GGTAATAGAC AAACATCCTG TCCGGTTTTT TGTACATAAG AGGCCCCATG TGGATTTCT420 CCTGGAAGTG GTGAGCCAGT GGTACGAGCT GGTGGTGTTT ACAGCAAGCA TGGAGATCT480 TGGCTCTGCT GTGGCAGATA AACTGGACAA TAGCAGAAGC ATTCTTAAGA GGAGATATT540 CAGACAGCAC TGCACTTTGG AGTTGGGCAG CTACATCAAG GACCTCTCTG TGGTCCACA600 TGACCTCTCC AGCATTGTGA TCCTGGATAA CTCCCCAGGG GCTTACAGGA GCCATCCAG660 CAATGCCATC CCCATCAAAT CCTGGTTCAG TGACCCCAGC GACACAGCCC TTCTCAACC720 GCTCCCAATG CTGGATGCCC TCAGGTTCAC CGCTGATGTT CGTTCCGTGC TGAGCCGAA780 CCTTCACCAA CATCGGCTCT GGTGACAGCT GCTCCCCCTC CACCTGAGTT GGGGTGGGG840 GGAAAGGGAG GGCGAGCCCT TGGGATGCCG TCTGATGCCC TGTCCAATGT GAGGACTGC900 TGGGCAGGGT CTGCCCCTCC CACCCCTCTC TGCCCTGGGA GCCCTACACT CCACTTGGA960 TCTGGATGGA CACATGGGCC AGGGGCTCTG AAGCAGCCTC ACTCTTAACT TCGTGTTC1020 ACTCCATGGA AACCCCAGAC TGGGACACAG GCGGAAGCCT AGGAGAGCCG AATCAGTG1080 TGTGAAGAGG CAGGACTGGC CAGAGTGACA GACATACGGT GATCCAGGAG GCTCAAAG1140 AAGCCAAGTC AGCTTTGTTG TGATTTGATT TTTTTTAAAA AACTCTTGTA CAAAACTG1200 CTAATTCTTC ACTCCTGCTC CAAGGGCTGG GCTGTGGGTG GGATACTGGG ATTTTGGG1260 ACTGGATTTT CCCTAAATTT GTCCCCCCTT TACTCTCCCT CTATTTTTCT CTCCTTAG1320 TCCCTCAGAC CTGTAACCAG CTTTGTGTCT TTTTTCCTTT TCTCTCTTTT AAACCATG1380 TTATAACTTT GAAACCAAAA AAAAAA 1406 2028 base pairs nucleic acid single linear UTRSNOT05 1568361 73 GNANANNACC CTGCNGGNNG CCATTCACCG ACCCTGCCCA NACAGCCGTC ACCCTCGANT 60 CCTGGCTGAN TCTNTTCCTG GCAGTTCCCC TTATNANGGT TACAACTATG GCTCCTTTN 120 CAATGTNTCT NTATCTACCG ATGGTCTGGT TNACAGCNCT GGCACTGGGG ACCTCTCTT 180 CGGTTACCAG GGCCGCTCCT TTGAACCTGT AGGTACTCGG CCCCGAGTGG ACTCCATGA 240 CTCTGTGGAG GAGGATGACT ACGACACATT GACCGACATC GATTCCGACA AGAATGTCA 300 TCGCACCAAG CAATACCTCT ATGTGGCTGA CCTGGCACGG AAGGACAAGC GTGTTCTGC 360 GAAAAAGTAC CAGATCTACT TCTGGAACAT TGCCACCATT GCTGTCTTCT ATGCCCTTC 420 TGTGGTGCAG CTGGTGATCA CCTACCAGAC GGTGGTGAAT GTCACAGGGA ATCAGGACA 480 CTGCTACTAC AACTTCCTCT GCGCCCACCC ACTGGGCAAT CTCAGCGCCT TCAACAACA 540 CCTCAGCAAC CTGGGGTACA TCCTGCTGGG GCTGCTTTTC CTGCTCATCA TCCTGCAAC 600 GGAGATCAAC CACAACCGGG CCCTGCTGCG CAATGACCTC TGTGCCCTGG AATGTGGGA 660 CCCCAAACAC TTTGGGCTTT TCTACGCCAT GGGCACAGCC CTGATGATGG AGGGGCTGC 720 CAGTGCTTGC TATCATGTGT GCCCCAACTA TACCAATTTC CAGTTTGACA CATCGTTCA 780 GTACATGATC GCCGGACTCT GCATGCTGAA GCTCTACCAG AAGCGGCACC CGGACATCA 840 CGCCAGCGCC TACAGTGCCT ACGCCTGCCT GGCCATTGTC ATCTTCTTNT CTGTGCTGG 900 CGTGGTCTTT GGCAAAGGGA ACACGGCGTT CTGGATCGTC TTCTCCATCA TTCACATCA 960 CGCCACCCTG CTCCTCAGCA CGCAGCTCTA TTACATGGGC CGGTGGAAAC TGGACTCG 1020 GATCTTCCGC CGCATCCTCC ACGTGCTCTA CACAGACTGC ATCCGGCAGT GCAGCGGG 1080 GCTCTACGTG GACCGCATGG TGCTGCTGGT CATGGGCAAC GTCATCAACT GGTCGCTG 1140 TGCCTATGGG CTTATCATGC GCCCCAATGA TTTCGCTTCC TACTTGTTGG CCATTGGC 1200 CTGCAACCTG CTCCTTTACT TCGCCTTCTA CATCATCATG AAGCTCCGGA GTGGGGAG 1260 GATCAAGCTC ATCCCCCTGC TCTGCATCGT TTGCACCTCC GTGGTCTGGG GCTTCGCG 1320 CTTCTTCTTC TTCCAGGGAC TCAGCACCTG GCAGAAAACC CCTGCAGAGT CGAGGGAG 1380 CAACCGGGAC TGCATCCTCC TCGACTTCTT TGACGACCAC GACATCTGGC ACTTCCTC 1440 CTCCATCGCC ATGTTCGGGT CCTTCCTGGT GTTGCTGACA CTGGATGACG ACCTGGAT 1500 TGTGCAGCGG GACAAGATCT ATGTCTTCTA GCAGGAGCTG GGCCCTTCGC TTCACCTC 1560 GGGGCCCTGA GCTCCTTTGT GTCATAGACC GGTCACTCTG TCGTGCTGTG GGGATGAG 1620 CCAGCACCGC TGCCCAGCAC TGGATGGCAG CAGGACAGCC AGGTCTAGCT TAGGCTTG 1680 CTGGGACAGC CATGGGGTGG CATGGAACCT TGCAGCTGCC CTCTGCCGAG GAGCAGGC 1740 GCTCCCCTGG AACCCCCAGA TGTTGGCCAA ATTGCTGCTT TCTTCTCAGT GTTGGGGC 1800 TCCATGGGCC CCTGTCCTTT GGCTCTCCAT TTGTCCCTTT GCAAGAGGAA GGATGGAA 1860 GACACCCTCC CCATTTCATG CCTTGCATTT TGCCCGTCCT CCTCCCCACA ATGCCCCA 1920 CTGGGACCTA AGGCCTCTTT TTCCTCCCAT ACTCCCACTC CAGGGCCTAG TCTGGGGC 1980 GAATCTCTGT CCTGTATCAG GGCCCCAGTT CTCTTTGGGC TGTCCCTG 2028 1380 base pairs nucleic acid single linear LNODNOT03 1572888 74 CTCGAGCCGA ATTCGGCTCG AGCGGCGCTC CTGCCTCCCT GCAGGGAGCT GCTTATGGGA 60 CACCGCTTCC TGCGCGGCCT CTTAACGCTG CTGCTGCCGC CGCCACCCCT GTATACCCG 120 CACCGCATGC TCGGTCCAGA GTCCGTCCCG CCCCCAAAAC GATCCCGCAG CAAACTCAT 180 GCACCGCCCC GAATCGGGAC GCACAATGGC ACCTTCCACT GCGACGAGGC ACTGGCATG 240 GCACTGCTTC GCCTCCTGCC GGAGTACCGG GATGCAGAGA TTGTGCGGAC CCGGGATCC 300 GAAAAACTCG CTTCCTGTGA CATCGTGGTG GACGTGGGGG GCGAGTACGA CCCTCGGAG 360 CACCGATATG ACCATCACCA GAGGTCTTTC ACAGAGACCA TGAGCTCCCT GTCCCCTGG 420 AAGCCGTGGC AGACCAAGCT GAGCAGTGCG GGACTCATCT ATCTGCACTT CGGGCACAA 480 CTGCTGGCCC AGTTGCTGGG CACTAGTGAA GAGGACAGCA TGGTGGGCAC CCTCTATGA 540 AAGATGTATG AGAACTTTGT GGAGGAGGTG GATGCTGTGG ACAATGGGAT CTCCCAGTG 600 GCAGAGGGGG AGCCTCGATA TGCACTGACC ACTACCCTGA GTGCACGAGT TGCTCGACT 660 AATCCTACCT GGAACCACCC CGACCAAGAC ACTGAGGCAG GGTTCAAGCG TGCAATGGA 720 CTGGTTCAAG AGGAGTTTCT GCAGAGATTA GATTTCTACC AACACAGCTG GCTGCCAGC 780 CGGGCCTTGG TGGAAGAGGC CCTTGCCCAG CGATTCCAGG TGGACCCAAG TGGAGAGAT 840 GTGGAACTGG CGAAAGGTGC ATGTCCCTGG AAGGAGCATC TCTACCACCT GGAATCTGG 900 CTGTCCCCTC CAGTGGCCAT CTTCTTTGTT ATCTACACTG ACCAGGCTGG ACAGTGGCG 960 ATACAGTGTG TGCCCAAGGA GCCCCACTCA TTCCAAAGCC GGCTGCCCCT GCCAGAGC 1020 TGGCGGGGTC TTCGGGACGA GGCCCTGGAC CAGGTCAGTG GGATCCCTGG CTGCATCT 1080 GTCCATGCAA GCGGCTTCAT TGGCGGTCAC CGCACCCGAG AGGGTGCCTT GAGCATGG 1140 CGTGCCACCT TGGCCCAGCG CTCATACCTC CCACAAATCT CCTAGTCTAA TAAAACCT 1200 CATCTCATAC TGACAAAAAA AAAATGGTAG CTCGGGGGGC GTTAGGATAG ATCTTTAG 1260 CCGGTGGGGT TTCTAAGCTG GAGAAGTGTT AGAAAAAAAG GGCGGCCGCC CATCAAAT 1320 GTTCTCTGCC CCGGGATTTT TTTTCGGGCC GGTACCCTTG GGGGGACCAA ATTTCCCC 1380 2028 base pairs nucleic acid single linear LNODNOT03 1573677 75 CAAAAGGACA AGATAATAAA GTACAAAATG GTTCGTTACA TCAGAAGGAT ACAGTTCATG 60 ACAATGACTT TGAGCCCTAC CTTACTGGAC AGTCAAATCA GAGTAACAGT TACCCCTCA 120 TGAGCGACCC CTACCTGTCC AGCTATTACC CGCCGTCCAT TGGATTTCCT TACTCCCTC 180 ATGAGGCTCC GTGGTCTACT GCAGGGGACC CTCCGATTCC ATACCTCACC ACCTACGGA 240 AGCTCAGTAA CGGAGACCAT CATTTTATGC ACGATGCTGT TTTTGGGCAG CCTGGGGGC 300 TGGGGAACAA CATCTATCAG CACAGGTTCA ATTTTTTCCC TGAAAACCCT GCGTTCTCA 360 CATGGGGGAC AAGTGGGTCT CAAGGTCAGC AGACCCAGAG CTCAGCCTCT CCCAGCACA 420 CCCCCAGCTT TGGCTCAACC GCAGTATCAG AGCCCTCAGC AGCCACCCCA GACCCGCTG 480 GTTGCCCCAC GCAACAGAAA CGCGGCGTTT GGGCAGAGCG GAGGGGCTGG CAGCGATAG 540 AACTCTCCTG GAAACGTCCA GCCTAATTCT GCCCCCAGCG TCGAATCCCA CCCCGTCCT 600 GAAAAACTGA AGGCTGCTCA CAGCTACAAC CCGAAAGAGT TTGAGTGGAA TCTGAAAAG 660 GGGCGTGTGT TCATCATCAA GAGCTACTCT GAGGACGACA TCCACCGCTC CATTAAGTA 720 TCCATCTGGT GTAGCACAGA GCACGGCAAC AAGCGCCTGG ACAGCGCCTT CCGCTGCAT 780 AGCAGCAAGG GGCCCGTCTA CCTGCTCTTC AGCGTCAATG GGAGTGGGCA TTTTTGTGG 840 GTGGCCGAGA TGAAGTCCCC CGTGGACTAC GGCACCAGTG CCGGGGTCTG GTCTCAGGA 900 AAGTGGAAGG GGAAGTTTGA TGTCCAGTGG ATTTTTGTTA AGGATGTACC CAATAACCA 960 CTCCGGCACA TCAGGCTGGA GAATAACGAC AACAAACCGG TCACAAACTC CCGGGACA 1020 CAGGAGGTGC CCTTAGAAAA AGCCAAGCAA GTGCTGAAAA TTATCAGTTC CTACAAGC 1080 ACAACCTCCA TCTTCGACGA CTTTGCTCAC TACGAGAAGC GCCAGAGGAG GAGGAGGT 1140 TGCGCAAGGA ACGGCAGAGT CGAAACAAAC AATGAGGGCG AACCAGTTTC TTACATGT 1200 TAACGTTTGA CTTTGAAAAC AGTTTAAAAC ACGTGTGCTT GGTCAGCTCC AGTGTGTC 1260 CCCGTGCGGG GGTTGAGTGT TGCATCTTTG CCTTTCTTGT CGTTGATTTT TGCCCAGA 1320 GATCTGCATT TATTTGTACT TTTTCTATGT ATTATAATCC TGTAGAAGTC ACTAATAA 1380 GAGTATTTTT TTTTGTCAGC TTATCAATCA GACTGATCTA ATGTGAAATG TAAGTATC 1440 TAAAAACAAA GCATCTATTT TGGCAGAAAT TGTGTTCTTA AATTCAGTCA TTTGATAT 1500 TGTGAGACTT CATATTTCTC ATCCCTTTAT TGCTTTTTAG CAAACATAAG AAACCATG 1560 TCATTTTGTC ATTTAGAGTA TTCTGATAAA ATCTCTTGAA AATACTGAAA TCAAAAGG 1620 AATGATTTTT TGTTCATTCT GATTTGTCAT TTTATTATCT GTTATCGGTC TAAAGTGC 1680 ATTTACCCAT TTGATTTTTC TGCTAGACAG ATAACTTTTA ATTTTTCAAA TTTGGCAG 1740 ACTTTTTTTT TTTTTTTGAA AATCTTTCCT TCCAGATCTG TTGCCCACTG AACAGCCA 1800 CGTCCCTCAC TGTCCTGGTG TCCGATTGGG CTGGATGGTG TTGGGGCATG ATGTGTGG 1860 GAACTGGAAG GTGCTTTAGG TCTGGTTCAG GGTCGGGCAT TCTTTGTTGT TTGCACAT 1920 TTTTAAATTT TACACCTTTT CTTAAGAATT CTAATGCCGT CTTAAGTTTT TATACCAA 1980 ATGCTGAGCT TTAAGTGTAG GATCTGGTAG TACAGACAGT GTGATGGA 2028 1170 base pairs nucleic acid single linear LNODNOT03 1574624 76 TCCGCACTAG GCCCGCCCCA ATTTGCGTGT TTTTACCGTG CAGAGGGAGG GATTTAGAGT 60 TAGCCTTTGA TTGGTCAGCT TGACTGGCGA CCTTTCCCCT CTGCGACAGT TTCCCGAGG 120 ACCTAGTGTC TGAGCGGCAC AGACGAGATC TCGATCGAAG GCGAGATGGC GGACGTGCT 180 GATCTTCACG AGGCTGGGGG CGAAGATTTC GCCATGGATG AGGATGGGGA CGAGAGCAT 240 CACAAACTGA AAGAAAAAGC GAAGAAACGG AAGGGTCGCG GCTTTGGCTC CGAAGAGGG 300 TCCCGAGCGC GGATGCGTGA GGATTATGAC AGCGTGGAGC AGGATGGCGA TGAACCCGG 360 CCACAACGCT CTGTTGAAGG CTGGATTCTC TTTGTAACTG GAGTCCATGA GGAAGCCAC 420 GAAGAAGACA TACACGACAA ATTCGCAGAA TATGGGGAAA TTAAAAACAT TCATCTCAA 480 CTCGACAGGC GAACAGGATA TCTGAAGGGG TATACTCTAG TTGAATATGA AACATACAA 540 GAAGCCCAGG CTGCTATGGA GGGACTCAAT GGCCAGGATT TGATGGGACA GCCCATCAG 600 GTTGACTGGT GTTTTGTTCG GGGTCCACCA AAAGGCAAGA GGAGAGGTGG CCGAAGACG 660 AGCAGAAGTC CAGACCGGAG ACGTCGCTGA CAGGTCCTCT GTTGTCCAGG TGTTCTCTT 720 AAGATTCCAT TTGACCATGC AGCCTTGGAC AAATAGGACT GGGGTGGAAC TTGCTGTGT 780 TATATTTAAT CTCTTACCGT ATATGCGTAG TATTTGAGTT GCGAATAAAT GTTCCATTT 840 TGTTTTCTAC ATTTAATGTT ACTTTCCTGT TCCCAAAATT GAAAGTTCTA AAGCATAGC 900 AGGCTGTATG GATCATTTGG AAAGATACCT TCTAGGGACT GAACCCCAAG TANTTCCTT 960 TNTTCCCTTT TCCGAAAATA ATCCCTGCTG TGTTTACCCG GGTGTGATTG CCCCTGAT 1020 TATCCCACTG CCGTCTCCAA ACTTTTGGGC TTCATCCTTC TTTCTGCCTC CTTGTCTC 1080 ATTTTTTGGG ATAATAGGCC CTTCTCCCCT CCCCATCAAA TATTTTATTA TTTTTTTC 1140 AAAAGGTTTT TCCGTTTTTC ACGGGGCCCT 1170 1107 base pairs nucleic acid single linear LNODNOT03 1577239 77 CCGAGCGCGG CCCCTGGGTT CGAACACGGC ACCCGCACTG CGCGTCATGG TGCAGGCCTG 60 GTATATGGAC GACGCCCCGG GCGACCCGCG GCAACCCCAC CGCCCCGACC CCGGCCGCC 120 AGTGGGCCTG GAGCAGCTGC GGCGGCTCGG GGTGCTCTAC TGGAAGCTGG ATGCTGACA 180 ATATGAGAAT GATCCAGAAT TAGAAAAGAT CCGAAGAGAG AGGAACTACT CCTGGATGG 240 CATCATAACC ATATGCAAAG ATAAACTACC AAATTATGAA GAAAAGATTA AGATGTTCT 300 CGAGGAGCAT TTGCACTTGG ACGATGAGAT CCGCTACATC CTGGATGGCA GTGGGTACT 360 CGACGTGAGG GACAAGGAGG ACCAGTGGAT CCGGATCTTC ATGGAGAAGG GAGACATGG 420 GACGCTCCCC GCGGGGATCT ATCACCGCTT CACGGTGGAC GAGAAGAACT ACACGAAGG 480 CATGCGGCTG TTTGTGGGAG AACCGGTGTG GACAGCGTAC AACCGGCCCG CTGACCATT 540 TGAAGCCCGC GGGCAGTACG TGAAATTTCT GGCACAGACC GCCTAGCAGT GCTGCCTGG 600 AACTAACACG CGCCTCGTAA AGGTCCCCAA TGTAATGACT GAGCAGAAAA TCAATCACT 660 TCTCTTTGCT TTTAGAGGAT AGCCTTGAGG CTAGATTATC TTTCCTTTGT AAGATTATT 720 GATCAGAATA TTTTGTAATG AAAGGATCTA GAAAGCAACT TGGAAGTGTA AAGAGTCAC 780 TTCATTTTCT GTAACTCAAT CAAGACTGGT GGGTCCATGG CCCTGTGTTA GTTCATGCA 840 TCAGTTGAGT CCCAAATGAA AGTTTCATCT CCCGAAATGC AGTTCCTTAG ATGCCCATC 900 GGACGTGATG CCGCGCCTGC CGTGTAAGAA GGTGCAATCC TAGATAACAC AGCTAGCCA 960 ATAGAAGACA CTTTTTTCTC CAAAATGATG CCTTGGGGTG GGGAGTGGTA GGGGGAAG 1020 CTCCCACCCT AAGGGGCACA CACTGAGTTG CTTATGCCAC TTCCTTGTTC AAAATAAA 1080 AACTGCCTTA ATCTTATACT CATGGCT 1107 1075 base pairs nucleic acid single linear BLADNOT03 1598203 78 CGGTAACCAG CCCTGGGAAG CCCGCAAGAG GCCTCAGCGG TGGCCGTCCG AGAGCCGAGA 60 GGTGAGGGTG CCCCCGCCTC ACCTGCAGAG GGGCCGTTCC GGGCTCGAAC CCGGCACCT 120 CCGGAAAATG GCGGCTGCCA GGCCCAGCCT GGGCCGAGTC CTCCCAGGAT CCTCTGTCC 180 GTTCCTGTGT GACATGCAGG AGAAGTTCCG CCACAACATC GCCTACTTCC CACAGATCG 240 CTCAGTGGCT GCCCGCATGC TCAAGGTGGC CCGGCTGCTT GAGGTGCCAG TCATGCTGA 300 GGAGCAGTAC CCACAAGGCC TGGGCCCCAC GGTGCCCGAG CTGGGGACTG AGGGCCTTC 360 GCCGCTGGCC AAGACCTGCT TCAGCATGGT GCCTGCCCTG CAGCAGGAGC TGGACAGTC 420 GCCCCAGCTG CGCTCTGTGC TGCTCTGTGG CATTGAGGCA CAGGCCTGCA TCTTGAACA 480 GACCCTGGAC CTCCTAGACC GGGGGCTGCA GGTCCATGTG GTGGTGGACG CCTGCTCCT 540 ACGCAGCCAG GTGGACCGGC TGGTGGCTCT GGCCCGCATG AGACAGAGTG GTGCCTTCC 600 CTCCACCAGC GAAGGGCTCA TTCTGCAGCT TGTGGGCGAT GCCGTCCACC CCCAGTTCA 660 GGAGATCCAG AAACTCATCA AGGAGCCCGC CCCAGACAGC GGACTGCTGG GCCTCTTCC 720 AGGCCAGAAC TCCCTCCTCC ACTGAACTCC AACCCTGCCT TGAGGGAAGA CCACCCTCC 780 GTCACCCGGA CCTCAGTGGA AGCCCGTTCC CCCCATCCCT GGATCCCAAG AGTGGTGCG 840 TCCACCAGGA GTGCCGCCCC CTTGTGGGGG GGGGCAGGGT GCTGCCTTCC CATTGGACA 900 CTGCTCCCGG AAATGCAAAT GAGACTCCTG GAAACTGGGT GGGAATTGGC TGAGCCAAG 960 TGGAGGCGGG GCTCGGCCCC GGGCCACTTC ACGGGGCGGG AAGGGGAGGG GAAGAAGA 1020 CTCAGACTGT GGGACACGGA CTCGCAGAAT AAACATATAT GTGGCAAAAA AAAAA 1075 1830 base pairs nucleic acid single linear BLADNOT03 1600438 79 GTCAATGTGT CTGTCCTTCA CTCCTCCATT GTCTGCCGCC ACTGCTGCTG CTGCTGCTGC 60 TGCCGCTGCT GCTGCACGAA TCGTCGCAGC CCCCAGCCTT GCGCGTCGTC GCTACCTCC 120 CGGACAGAAA TTTTATGAAT AAGCATCAGA AGCCAGTGCT AACAGGCCAG CGGTTCAAA 180 CTCGGAAAAG GGATGAAAAA GAGAAATTCG AACCCACAGT CTTCAGGGAT ACACTTGTC 240 AGGGGCTTAA TGAGGCTGGT GATGACCTTG AAGCTGTAGC CAAATTTCTG GACTCTACA 300 GCTCAAGATT AGATTATCGT CGCTATGCAG ACACACTCTT CGATATCCTG GTGGCTGGC 360 GTATGCTTGC CCCTGGAGGA ACGCGCATAG ATGATGGTGA CAAGACCAAG ATGACCAAC 420 ACTGTGTGTT TTCAGCAAAT GAAGATCATG AAACCATCCG AAACTATGCT CAGGTCTTC 480 ATAAACTCAT CAGGAGATAT AAGTATTTGG AGAAGGCATT TGAAGATGAA ATGAAAAAG 540 TTCTCCTCTT CCTTAAAGCC TTTTCCGAAA CAGAGCAGAC AAAGTTGGCG ATGCTGTCG 600 GGATTCTGCT GGGCAATGGC ACCCTGCCCG CCACCATCCT CACCAGTCTC TTCACCGAC 660 GCTTAGTCAA AGAAGGCATT GCGGCCTCAT TTGCTGTCAA GCTTTTCAAA GCATGGATG 720 CAGAAAAAGA TGCCAACTCT GTTACCTCGT CTTTGAGAAA AGCCAACTTA GACAAGAGG 780 TGCTTGAACT CTTTCCAGTT AACAGACAGA GTGTGGATCA TTTTGCTAAA TACTTCACT 840 ACGCAGGTCT TAAGGAGCTT TCCGACTTCC TCCGAGTCCA GCAGTCCCTG GGCACCAGG 900 AGGAACTGCA GAAGGAGCTC CAGGAGCGTC TTTCTCAGGA ATGCCCGATC AAGGAGGTG 960 TGCTTTATGT CAAAGAAGAA ATGAAGAGGA ATGATCTTCC AGAAACAGCA GTGATTGG 1020 TTCTGTGGAC ATGTATAATG AACGCTGTTG AGTGGAACAA GAAGGAAGAA CTTGTTGC 1080 AGCAGGCTCT GAAGCACCTG AAGCAATATG CTCCCCTGCT GGCCGTGTTC AGCTCCCA 1140 GCCAGTCAGA GCTGATCCTC CTCCAGAAGG TTCAGGAATA CTGCTACGAC AACATCCA 1200 TCATGAAAGC CTTTCAGAAG ATTGTGGTTC TCTTTTATAA AGCTGATGTT CTGAGCGA 1260 AAGCAATACT GAAATGGTAT AAGGAAGCAC ATGTTGCTAA AGGCAAAAGT GTTTTTCT 1320 ACCAGATGAA GAAATTTGTT GAGTGGTTAC AAAATGCAGA AGAAGAATCC GAATCGGA 1380 GTGAGGAAAA TTAAATGGCT CAACAAGCAC AATACCTAGG TTACCACACA CCACTTTT 1440 ATTGGGAATG CTGAACCATT TGAGAAGAGA AACTTGGCTT CTGTTTTCGC AAAGGAAA 1500 AAAAATAGGA TAGGCTTCCC TTGTGCAGAG GGAGAAATGG TTTTGTTTTT GTTTTGTT 1560 TAAATGGAGC CCTGAGGCAT CAGCTATTAT ACTTGGGACT CTACCTCTCA CTCACTAT 1620 GCTAACTTAA AGCCATTCAA CAAGGAGTCA AGTAGATCTG AAATTAAATA CTCAACAG 1680 TCCTCCTTTT TTAGCTGTAT TTTTCAGGTA CTGTGTGGTG ACCGCCCCAC TGGTGTCT 1740 TACAGGCCAC TTTGGTAGTT GTGTATCTGC TCATGTATGT GATTTGACAA ACCAGTTT 1800 TAAAATAAAT GGCTTTTTAA GAAAAAAAAA 1830 1330 base pairs nucleic acid single linear BLADNOT03 1600518 80 CCGGCAGCCA TCCCCGCGGT GCTGACATCC CGGTTGTTCT TCTGTGCCGG GGGTCTTCCT 60 GCTGTCATGA AGGACGTACC GGGCTTCCTA CAGCAGAGCC AGAGCTCCGG GCCCGGGCA 120 CCCGCTGTGT GGCACCGTCT GGAGGAGCTC TACACGAAGA AGTTGTGGCA TCAGCTGAC 180 CTTCAGGTGC TTGATTTTGT GCAGGATCCG TGCTTTGCCC AAGGAGATGG TCTCATTAA 240 CTTTATGAAA ACTTTATCAG TGAATTTGAA CACAGGGTGA ATCCTTTGTC CCTCGTGGA 300 ATCATTCTTC ATGTAGTTAG ACAGATGACT GATCCTAATG TGGCTCTTAC TTTTCTGGA 360 AAGACTCGTG AGAAGGTGAA AAGTAGTGAT GAGGCAGTGA TCCTGTGTAA AACAGCAAT 420 GGAGCTCTAA AATTAAACAT CGGGGACCTA CAGGTTACAA AGGAAACAAT TGAAGATGT 480 GAAGAAATGC TCAACAACCT TCCTGGTGTG ACATCGGTTC ACAGTCGTTT CTATGATCT 540 TCCAGTAAAT ACTATCAAAC AATCGGAAAC CACGCGTCCT ACTACAAAGA TGCTCTGCG 600 TTTTTGGGCT GTGTTGACAT CAAGGATCTA CCAGTGTCTG AGCAGCAGGA GAGAGCCTT 660 ACGCTGGGGC TAGCAGGACT TCTCGGCGAG GGAGTTTTTA ACTTTGGAGA ACTCCTCAT 720 CACCCTGTGC TGGAGTCCCT GAGGAATACT GACCGGCAGT GGCTGATTGA CACCCTCTA 780 GCCTTCAACA GTGGCAACGT AGAGCGGTTC CAGACTCTGA AGACTGCCTG GGGCCAGCA 840 CCTGATTTAG CAGCTAATGA AGCCCAGCTT CTGAGGAAAA TTCAGTTGTT GTGCCTCAT 900 GAGATGACTT TCACACGACC TGCCAATCAC AGACAACTCA CTTTTGAAGA AATTGCCAA 960 AGTGCTAAAA TCACAGTGAA TGAGGTGGAG CTTCTGGTGA TGAAGGCCCT TTCGGTGG 1020 CTGGTGAAAG GCAGTATAGA CGAGGTGGAC AAACGAGTCC ACATGACCTG GGTGCAGC 1080 CGAGTGTTGG ATTTGCAACA GATCAAGGGA ATGAAGGACC GCCTGGAGTT CTGGTGCA 1140 GATGTGAAGA GCATGGAGAT GCTGGTGGAG CACCAGGCCC ATGACATCCT CACCTAGG 1200 CCCCTGGTTC CCCGTCGTGT CTCCTTTGAC TCACCTGAGA GAGGCGTTTG CAGCCAAT 1260 AGCTGGCTGC TCAGACGGTC GACATTGAAT TTGGGTGGGG GTTGGGATCC TGTCTGAA 1320 ACAGAATGTT 1330 1152 base pairs nucleic acid single linear BLADNOT03 1602473 81 CGAGCCGGCG CACCGTACGC TGGGACGTGT GGTTTCAGCT CGTGCGCCTC CCCGTGGGTT 60 TGCGACGTTT AGCGACTATT GCGCCTGCGC CAGCGCCGGC TGCGAGACTG GGGCCGTGG 120 TGCTGGTCCC GGGTGATGCT AGGCGGCTCC CTGGGCTCCA GGCTGTTGCG GGGTGTAGG 180 GGGAGTCACG GACGGTTCGG GGCCCGAGGT GTCCGCGAAG GTGGCGCAGC CATGGCGGC 240 GGGGAGAGCA TGGCTCAGCG GATGGTCTGG GTGGACCTGG AGATGACAGG ATTGGACAT 300 GAGAAGGACC AGATTATTGA GATGGCCTGT CTGATAACTG ACTCTGATCT CAACATTTT 360 GCTGAAGGTC CTAACCTGAT TATAAAACAA CCAGATGAGT TGCTGGACAG CATGTCAGA 420 TGGTGTAAGG AGCATCACGG GAAGTCTGGC CTTACCAAGG CAGTGAAGGA GAGTACAAT 480 ACATTGCAGC AGGCAGAGTA TGAATTTCTG TCCTTTGTAC GACAGCAGAC TCCTCCAGG 540 CTCTGTCCAC TTGCAGGAAA TTCAGTTCAT GAAGATAAGA AGTTTCTTGA CAAATACAT 600 CCCCAGTTCA TGAAACATCT TCATTATAGA ATAATTGATG TGAGCACTGT TAAAGAACT 660 TGCAGACGCT GGTATCCAGA AGAATATGAA TTTGCACCAA AGAAGGCTGC TTCTCATAG 720 GCACTTGATG ACATTAGTGA AAGCATCAAA GAGCTTCAGT TTTACCGAAA TAACATCTT 780 AAGAAAAAAA TAGATGAAAA GAAGAGGAAA ATTATAGAAA ATGGGGAAAA TGAGAAGAC 840 GTGAGTTGAT GCCAGTTATC ATGCTGCCAC TACATCGTTA TCTGGAGGCA ACTTCTGGT 900 GTTTTTTTTT CTCACGCTGA TGGCTTGGCA GAGCACCTTC GGTTAACTTG CATCTCCAG 960 TTGATTACTC AAGCAGACAG CACACGAAAT ACTATTTTTC TCCTAATATG CTGTTTCC 1020 TATGACACAG CAGCTCCTTT GTAAGTACCA GGTCATGTCC ATCCCTTGGT ACATATAT 1080 ATTTGCTTTT AAACCATTTC TTTTGTTTAA ATAAATAAAT AAGTAAATAA AGCTAGTT 1140 ATTGAAATGC AA 1152 566 base pairs nucleic acid single linear LUNGNOT15 1605720 82 GTTCTCCGCC CCTGCCACTG GGCCATGGAG ACTGTGGCAC AGTAGACTGT AGTGTGAGGC 60 TCGCGGGGGC AGTGGCCATG GAGGCCGTGC TGAACGAGCT GGTGTCTGTG GAGGACCTG 120 TGAAGTTTGA AAAGAAATTT CAGTCTGAGA AGGCAGCAGG CTCGGTGTCC AAGAGCACG 180 AGTTTGAGTA CGCCTGGTGC CTGGTGCGGA GCAAGTACAA TGATGACATC CGTAAAGGC 240 TCGTGCTGCT CGAGGAGCTG CTGCCCAAAG GGAGCAAGGA GGAACAGCGG GATTACGTC 300 TCTACCTGGC CGTGGGGAAC TACCGGCTCA AGGAATACGA GAAGGCCTTA AAGTACGTC 360 GCGGGTTGCT GCAGACAGAG CCCCAGAACA ACCAGGCCAA GGAACTGGAG CGGCTCATT 420 ACAAGGCCAT GAAGAAAGAT GGACTCGTGG GCATGGCCAT CGTGGGAGGC ATGGCCCTG 480 GTGTGGCGGG ACTTGCCGGA CTCATCGGAC TTGCTGTGTC CAAGTCCAAA TTCTGAAGG 540 GACGCGGGAG CCCACGGAGA ACGCTC 566 745 base pairs nucleic acid single linear COLNTUT06 1610501 83 CGGAAGAGGT AGCTCACGCG ATAGAAACGT GTTCGCTGCC CAGAAGAAGG GAAGGCGCGA 60 GTGAGGAAAG GAGGTACTGT AGATGCCCTC CAAATCCTTG GTTATGGAAT ATTTGGCTC 120 TCCCAGTACA CTCGGCTTGG CTGTTGGAGT TGCTTGTGGC ATGTGCCTGG GCTGGAGCC 180 TCGAGTATGC TTTGGGATGC TCCCCAAAAG CAAGACGAGC AAGACACACA CAGATACTG 240 AAGTGAAGCA AGCATCTTGG GAGACAGCGG GGAGTACAAG ATGATTCTTG TGGTTCGAA 300 TGACTTAAAG ATGGGAAAAG GGAAAGTGGC TGCCCAGTGC TCTCATGCTG CTGTTTCAG 360 CTACAAGCAG ATTCAAAGAA GAAATCCTGA AATGCTCAAA CAATGGGAAT ACTGTGGCC 420 GCCCAAGGTG GTGGTCAAAG CTCCTGATGA AGAAACCCTG ATTGCATTAT TGGCCCATG 480 AAAAATGCTG GGACTGACTG TAAGTTTAAT TCAAGATGCT GGACGTACTC AGATTGCAC 540 AGGCTCTCAA ACTGTCCTAG GGATTGGGCC AGGACCAGCA GACCTAATTG ACAAAGTCA 600 TGGTCACCTA AAACTTTACT AGGTGGACTT TGATATGACA ACAACCCCTC CATCACAAG 660 GTTTGAAGCC TGTCAGATTC TAACAACAAA AGCTGAATTT CTTCACCCAA CTTAAATGT 720 CTTGAGATGA AAATAAAACC TATTA 745 909 base pairs nucleic acid single linear BLADNOT06 1720770 84 TGAGGGAGAC CGCGGCTCGG CCGTAGCGGA GCTGCGAGGT GGCAGGGCCC AGCCCCGAAC 60 CAGACAAGGG ACCCCTCAAG GAGCTTCATT CTAGCAGGAG AAAATTGAGA AGTAAACCA 120 AAAGTTACAG AATGTCTGAA GGGGACAGTG TGGGAGAATC CGTCCATGGG AAACCTTCG 180 TGGTGTACAG ATTTTTCACA AGACTTGGAC AGATTTATCA GTCCTGGCTA GACAAGTCC 240 CACCCTACAC GGCTGTGCGA TGGGTCGTGA CACTGGGCCT GAGCTTTGTC TACATGATT 300 GAGTTTACCT GCTGCAGGGT TGGTACATTG TGACCTATGC CTTGGGGATC TACCATCTA 360 ATCTTTTCAT AGCTTTTCTT TCTCCCAAAG TGGATCCTTC CTTAATGGAA GACTCAGAT 420 ACGGTCCTTC GCTACCCACC AAACAGAACG AGGAATTCCG CCCCTTCATT CGAAGGCTC 480 CAGAGTTTAA ATTTTGGCAT GCGGCTACCA AGGGCATCCT TGTGGCTATG GTCTGTACT 540 TCTTCGACGC TTTCAACGTC CCGGTGTTCT GGCCGATTCT GGTGATGTAC TTCATCATG 600 TCTTCTGTAT CACGATGAAG AGGCAAATCA AGCACATGAT TAAGTACCGG TACATCCCG 660 TCACACATGG GAAGAGAAGG TACAGAGGCA AGGAGGATGC CGGCAAGGCC TTCGCCAGC 720 AGAAGCGGGA CTGAGGCTGC CTCACGTGTT GCAAGAACAG TTTTGAGCCA TTGTTAACA 780 TGCCTTTTTT CTTCACATAA AGTAGTTGAT TACGAGGGAG TCAAATTTTC TTTTTAAAA 840 GGAGCTTCAA TGATTTGTAA CTGAAATATC AGGTTCTAGA AGAAACTGGC GCTTAAACC 900 AAAAAAAAA 909 2028 base pairs nucleic acid single linear BRAINON01 1832295 85 CCGGCGGGCG GGGGCTGAGG GGCTGCCATG GCGGCGGCGG GCCGGCTCCC GAGCTCCTGG 60 GCCCTCTTCT CGCCGCTCCT CGCAGGGCTT GCACTACTGG GAGTCGGGCC GGTCCCAGC 120 CGGGCGCTGC ACAACGTCAC GGCCGAGCTC TTTGGGGCCG AGGCCTGGGG CACCCTTGC 180 GCTTTCGGGG ACCTCAACTC CGACAAGCAG ACGGATCTCT TCGTGCTGCG GGAAAGAAA 240 GACTTAATCG TCTTTTTGGC AGACCAGAAT GCACCCTATT TTAAACCCAA AGTAAAGGT 300 TCTTTCAAGA ATCACAGTGC ATTGATAACA AGTGTAGTCC CTGGGGATTA TGATGGAGA 360 TCTCAAATGG ATGTCCTTCT GACATATCTT CCCAAAAATT ATGCCAAGAG TGAATTAGG 420 GCTGTTATCT TCTGGGGACA AAATCAAACA TTAGATCCTA ACAATATGAC CATACTCAA 480 AGGACTTTTC AAGATGAGCC ACTAATTATG GATTTCAATG GTGATCTAAT TCCTGATAT 540 TTTGGTATCA CAAATGAATC CAACCAGCCA CAGATACTAT TAGGAGGGAA TTTATCATG 600 CATCCAGCAT TGACCACTAC AAGTAAAATG CGAATTCCAC ATTCTCATGC ATTTATTGA 660 CTGACTGAAG ATTTTACAGC AGATTTATTC CTGACGACAT TGAATGCCAC CACTAGTAC 720 TTCCAGTTTG AAATATGGGA AAATTTGGAT GGAAACTTCT CTGTCAGTAC TATATTGGA 780 AAACCTCAAA ATATGATGGT GGTTGGACAG TCAGCATTTG CAGACTTTGA TGGAGATGG 840 CACATGGATC ATTTACTGCC AGGCTGTGAA GATAAAAATT GCCAAAAGAG TACCATCTA 900 TTAGTGAGAT CTGGGATGAA GCAGTGGGTT CCAGTCCTAC AAGATTTCAG CAATAAGGG 960 ACACTCTGGG GCTTTGTGCC ATTTGTGGAT GAACAGCAAC CAACTGAAAT ACCAATTC 1020 ATTACCCTTC ATATTGGAGA CTACAATATG GATGGCTATC CAGACGCTCT GGTCATAC 1080 AAGAACACAT CTGGAAGCAA CCAGCAGGCC TTTTTACTGG AGAACGTCCC TTGTAATA 1140 GCAAGCTGTG AAGAGGCGCG TCGAATGTTT AAAGTCTACT GGGAGCTGAC AGACCTAA 1200 CAAATTAAGG ATGCCATGGT TGCCACCTTC TTTGACATTT ACGAAGATGG AATCTTGG 1260 ATTGTAGTGC TAAGTAAAGG ATATACAAAG AATGATTTTG CCATTCATAC ACTAAAAA 1320 AACTTTGAAG CAGATGCTTA TTTTGTTAAA GTTATTGTTC TTAGTGGTCT GTGTTCTA 1380 GACTGTCCTC GTAAGATAAC GCCCTTTGGA GTGAATCAAC CTGGACCTTA TATCATGT 1440 ACAACTTTAG ATGCAAATGG GTATCTGAAA AATGGATCAG CTGGCCAACT CAGCCAAT 1500 GCACATTTAG CTCTCCAACT ACCATACAAC GTGCTTGGTT TAGGTCGGAG CGCAAATT 1560 CTTGACCATC TCTACGTTGG TATTCCCCGT CCATCTGGAG AAAAATCTAT ACGAAAAC 1620 GAGTGGACTG CAATCATTCC AAATTCCCAG CTAATTGTCA TTCCATACCC TCACAATG 1680 CCTCGAAGTT GGAGTGCCAA ACTGTATCTT ACACCAAGTA ATATTGTTCT GCTTACTG 1740 ATAGCTCTCA TCGGTGTCTG TGTTTTCATC TTGGCAATAA TTGGCATTTT ACATTGGC 1800 GAAAAGAAAG CAGATGATAG AGAAAAACGA CAAGAAGCCC ACCGGTTTCA TTTTGATG 1860 ATGTGACTTG CCTTTAATAT TACATAATGG AATGGCTGTT CACTTGATTA GTTGAAAC 1920 AAATTCTGGC TTGAAAAAAT AGGGGAGATT AAATATTATT TATAAATGAT GTATCCCA 1980 GTAATTATTG GAAAGTATTC AAATAAATAT GGTTTGAATA TGTCACAA 2028 372 base pairs nucleic acid single linear CORPNOT02 1990522 86 GCGAGCTCGG CTTCCTCAAC ATGGCTGCGC CCTTGTCAGT GGAGGTGGAG TTCGGAGGTG 60 GTGCGGAGCT CCTGTTTGAC GGTATTAAGA AACATCGAGT CACTTTGCCT GGACAGGAG 120 AACCCTGGGA CATCCGGAAC CTGCTCATCT GGATCAAGAA GAATTTGCTA AAAGAGCGG 180 CAGAGTTGTT CATCCAGGGA GACAGCGTGC GGCCAGGAAT TCTGGTGCTG ATTAACGAT 240 CCGACTGGGA GCTACTGGGT GAGCTGGACT ACCAGCTTCA GGACCAGGAC AGCGTCCTC 300 TCATCTCCAC TCTGCACGGC GGCTGAGGGC CCTTCTCTGG GGCTGGGCAA CCTTAGAGG 360 GAGAACGAAA AA 372 829 base pairs nucleic acid single linear BRAITUT02 2098087 87 CAGGCTCTGT ATCCGTGGCA GCGGCCGTGG CAGGCTGGCT GGGTACCGGC TGTCGCTGAC 60 CCAGGAGAAG CTGCCTGTCT ACATCAGCCT GGGCTGCAGC GCGCTGCCGC CGCGGGGCC 120 GCAGCCATGG CCAAGGACAT CCTGGGTGAA GCAGGGCTAC ACTTTGATGA ACTGAACAA 180 CTGAGGGTGT TGGACCCAGA GGTTACCCAG CAGACCATAG AGCTGAAGGA AGAGTGCAA 240 GACTTTGTGG ACAAAATTGG CCAGTTTCAG AAAATAGTTG GTGGTTTAAT TGAGCTTGT 300 GATCAACTTG CAAAAGAAGC AGAAAATGAA AAGATGAAGG CCATCGGTGC TCGGAACTT 360 CTCAAATCTA TAGCAAAGCA GAGAGAAGCT CAACAGCAGC AACTTCAAGC CCTAATAGC 420 GAAAAGAAAA TGCAGCTAGA AAGGTATCGG GTTGAATATG AAGCTTTGTG TAAAGTAGA 480 GCAGAACAAA ATGAATTTAT TGACCAATTT ATTTTTCAGA AATGAACTGA AAATTTCGC 540 TTTATAGTAG GAAGGCAAAA CAAAAAAAAG CCTCTCAAAA CCAAAAAAAC CTCTGTAGC 600 TTCCAGCGGC TTGACCAATG ACCTATGTCA CAAGAGGTGC GTGTAAGGAA TGCAGCCCC 660 TGGAACGTGC TATTCACGTC TGTGGGAGCC AGTTTTAACA TCAGTGCACA GCTGCTGCT 720 GTGGCCCTGC AGTGTACGTT CTCACCTCTT ATGCTTAGTT GGAACCCGAA CAAAAATAA 780 CTTTCATCCT TTTGTGTGTA ACTTCTCCAG AATACTAAAT AAAAAGTTC 829 1178 base pairs nucleic acid single linear BRAITUT03 2112230 88 CTTCCCGCCA GTCCCCTAAC CCTGAGGCTG CCGCGCGGCG GTCACTGCGC CGGGGTAGTG 60 GGCCCCAGTG TTGCGCTCTC TGGCCGTTCC TTACACTTTG CTTCAGGCTC CAGTGCAGG 120 GCGTAGTGGG ATATGGCCAA CTCGGGCTGC AAGGACGTCA CGGGTCCAGA TGAGGAGAG 180 TTTCTGTACT TTGCCTACGG CAGCAACCTG CTGACAGAGA GGATCCACCT CCGAAACCC 240 TCGGCGGCGT TCTTCTGTGT GGCCCGCCTG CAGGATTTTA AGCTTGACTT TGGCAATTC 300 CAAGGCAAAA CAAGTCAAAC TTGGCATGGA GGGATAGCCA CCATTTTTCA GAGTCCTGG 360 GATGAAGTGT GGGGAGTAGT ATGGAAAATG AACAAAAGCA ATTTAAATTC TCTGGATGA 420 CAAGAAGGGG TTAAAAGTGG AATGTATGTT GTAATAGAAG TTAAAGTTGC AACTCAAGA 480 GGAAAAGAAA TAACCTGTCG AAGTTATCTG ATGACAAATT ACGAAAGTGC TCCCCCATC 540 CCACAGTATA AAAAGATTAT TTGCATGGGT GCAAAAGAAA ATGGTTTGCC GCTGGAGTA 600 CAAGAGAAGT TAAAAGCAAT AGAACCAAAT GACTATACAG GAAAGGTCTC AGAAGAAAT 660 GAAGACATCA TCAAAAAGGG GGAAACACAA ACTCTTTAGA ACATAACAGA ATATATCTA 720 GGGTATTCTA TGTGCTAATA TAAAATATTT TTAACACTTG AGAACAGGGA TCTGGGGGA 780 CTCCACGTTT GATCCATTTT CAGCAGTGCT CTGAAGGAGT ATCTTACTTG GGTGATTCC 840 TGTTTTTAGA CTATAAAAAG AAACTGGGAT AGGAGTTAGA CAATTTAAAA GGGGTGTAT 900 AGGGCCTGAA ATATGTGACA AATGAATGTG AGTACCCCTT CTGTGAACAC TGAAAGCTA 960 TCTCTTGAAT TGATCTTAAG TGTCTCCTTG CTCTGGTAAA AGATAGATTT GTAGCTCA 1020 TGATGATGGT GCTGGTGAAT TGCTCTGCTC TGTCTGAGAT TTTTAAAAAT CAGCTTAA 1080 AGAGTAATCT GCAGACAATT GATAATAACA TTTTGAAAAT TGGAAAGATG GTATACTG 1140 TTTAGAGGAA TAAACGTATT TGTGGTTTAA AAAAAAAA 1178 748 base pairs nucleic acid single linear BRSTTUT02 2117050 89 CCCACGCGTC CGGCGACGGC GCGGACCTGG AGCTTCCGCG CGGTGGCTTC ACTCTCCTGT 60 AAAACGCTAG AGCGGCGAGT TGTTACCTGC GTCCTCTGAC CTGAGAGCGA AGGGGAAAG 120 GGCGAGATGA CTGACCGCTA CACCATCCAT AGCCAGCTGG AGCACCTGCA GTCCAAGTA 180 ATCGGCACGG GCCACGCCGA CACCACCAAG TGGGAGTGGC TGGTGAACCA ACACCGCGA 240 TCGTACTGCT CCTACATGGG CCACTTCGAC CTTCTCAACT ACTTCGCCAT TGCGGAGAA 300 GAGAGCAAAG CGCGAGTCCG CTTCAACTTG ATGGAAAAGA TGCTTCAGCC TTGTGGACC 360 CCAGCCGACA AGCCCGAGGA GAACTGAGAC TCTGCCTTAC CACCGCAGTG CGGGGGCAC 420 TCTCCCAGCG TTTCTCCGGT TTGCCAATCC TCTTAAGTAT TCCTGTCTCC AAAGGACCG 480 CTCTCCATGG CTCCTGCGCC TCGTGCTTTC CGCGTACAGA AGTGCTTGCC CGGGGAGTC 540 CGCCTGACCT GCCTTCATGT GGACCCTTAG AACAGCACTG GGAGACCAGC AGGACTCCT 600 AGAACTGTGC TGGTGGAGAG GTCCTAGAGC CGGCGAGCGT TTGAGAAGAG GGCATGGCG 660 TGGAGTGAGA TGGGATTTGG CGTCTCGTTT TTGGCTAATT GATTGTCATT GGCTTTTTC 720 ATAAAGTTTA GAAATCGTAA AAAAAAAA 748 1078 base pairs nucleic acid single linear SININOT01 2184712 90 GCAGGACGGA TTGGGCAAGG CTGGTCCCTG TGTGATGAGA CATCACCCTC CCAGGAGCAA 60 GGCGGAAGTC TGGAGGACGC TGAGGGGCGG AGGCGGGAGA GGCGAGCTCG CGATGAGTG 120 TCTCGGCAGG CTCTTCGGGA AGGGGAAGAA GGAGAAAGGG CCAACCCCTG AAGAAGCAA 180 ACAGAAACTG AAGGAGACAG AGAAGATACT GATCAAGAAA CAGGAATTTT TGGAGCAGA 240 GATTCAACAG GAGCTACAAA CAGCCAAGAA GTATGGGACC AAGAATAAGA GAGCTGCCC 300 ACAGGCTTTG CGGAGGAAGA AAAGATTCGA ACAGCAGCTG GCACAAACTG ACGGGACAT 360 ATCCACCCTG GAGTTTCAGC GTGAGGCCAT TGAGAATGCC ACTACCAATG CAGAAGTCC 420 TCGTACCATG GAGCTTGCTG CCCAAAGCAT GAAGAAGGCC TACCAGGACA TGGACATTG 480 CAAGGTAGAT GAACTGATGA CTGACATCAC GGAACAACAG GAGGTGGCCC AGCAGATCT 540 AGATGCCATT TCTCGGCCTA TGGGCTTTGG AGATGATGTG GATGAGGATG AACTGCTGG 600 GGAGCTAGAG GAGCTGGAGC AGGAGGAATT GGCCCAGGAG TTGTTAAATG TGGGCGACA 660 GGAAGAAGAA CCCTCAGTCA AATTGCCTAG TGTACCTTCT ACTCATCTGC CGGCAGGGC 720 AGCTCCCAAA GTGGATGAAG ATGAAGAAGC ACTAAAGCAG TTGGCTGAGT GGGTATCCT 780 ATAAATCTGG GCTTGTCTTC CTAATGCTAC CTTTGTTGGT CCTTTCTTCC TTAAGTGCC 840 AGTGCTGAGC TAAAGGAGGA TAACTTTTTG GGGAAGTCAT GCTGAGGGTG GTAGTGTGA 900 CCTGCCTGAA AAAAGGGTCT CTTACCCTCC CAGCCCTGGC TCAACTCTGA AGAAGGATC 960 TGCTACAGAA GGAGCCCTTG GGCTCCCTTC TCTTTGATAG CAGTTATAAT GCCCTTGT 1020 CCAATAAAAC TGGGCAGATG GAATCCTAGT GTCTATACTG CCTTGTCTAC CCCTGAAG 1078 1446 base pairs nucleic acid single linear BRAINON01 2290475 91 TGGGAGGCGG AGGCACAACT AAGAGCGACC TAGCATCGCA AAGCCGCCCT CGGGGCGCTC 60 ATGGCGGGAC GCTCCTGGGA AAGGCTTTAG CGCGGTGTCT CTCTCTCTGG CCTTGGCCT 120 TGTGACTATC AGGTCCTCGC GCTGCCGCGG CATCCAGGCG TTCAGAAACT CGTTTTCAT 180 TTCTTGGTTT CATCTTAATA CCAACGTCAT GTCTGGTTCT AATGGTTCCA AAGAAAATT 240 TCACAATAAG GCTCGGACGT CTCCTTACCC AGGTTCAAAA GTTGAACGAA GCCAGGTTC 300 TAATGAGAAA GTGGGCTGGC TTGTTGAGTG GCAAGACTAT AAGCCTGTGG AATACACTG 360 AGTCTCTGTC TTGGCTGGAC CCAGGTGGGC AGATCCTCAG ATCAGTGAAA GTAATTTTT 420 TCCCAAGTTT AACGAAAAGG ATGGGCATGT TGAGAGAAAG AGCAAGAATG GCCTGTATG 480 GATTGAAAAT GGAAGACCGA GAAATCCTGC AGGACGGACT GGACTGGTGG GCCGGGGGC 540 TTTGGGGCGA TGGGGCCCAA ATCACGCTGC AGATCCCATT ATAACCAGAT GGAAAAGGG 600 TAGCAGTGGA AATAAAATCA TGCATCCTGT TTCTGGGAAG CATATCTTAC AATTTGTTG 660 AATAAAAAGG AAAGACTGTG GAGAATGGGC AATCCCAGGG GGGATGGTGG ATCCAGGAG 720 GAAGATTAGT GCCACACTGA AAAGAGAATT TGGTGAGGAA GCTCTCAACT CCTTACAGA 780 AACCAGTGCT GAGAAGAGAG AAATAGAGGA AAAGTTGCAC AAACTCTTCA GCCAAGACC 840 CCTAGTGATA TATAAGGGAT ATGTTGATGA TCCTCGAAAC ACTGATAATG CATGGATGG 900 GACAGAAGCT GTGAACTACC ATGACGAAAC AGGTGAGATA ATGGATAATC TTATGCTAG 960 AGCTGGAGAT GATGCTGGAA AAGTGAAATG GGTGGACATC AATGATAAAC TGAAGCTT 1020 TGCCAGTCAC TCTCAATTCA TCAAACTTGT GGCTGAGAAA CGAGATGCAC ACTGGAGC 1080 GGACTCTGAA GCTGACTGCC ATGCGTTGTA GCTGATGGTC TCCGTGTAAG CCAAAGGC 1140 ACAGAGGAGC ATATACTGAA AAGAAGGCAG TATCACAGAA TTTATACTAT AAAAAGGG 1200 GGGTAGGCCA CTTGGCCTAT TTACTTTCAA AACAATTTGC ATTTAGAGTG TTTCGCAT 1260 GAATAACATG AGTAAGATGA ACTGGAACAC AAAATTTTCA GCTCTTTGGT CAAAAGGA 1320 ATAAGTAATC ATATTTTGTA TGTATTCGAT TTAAGCATGG CTTAAATTAA ATTTAAAC 1380 CTAATGCTCT TTGAAGAATC ATAATCAGAA TAAAGATAAA TTCTTGATCA GCTATAAA 1440 AAAAAA 1446 659 base pairs nucleic acid single linear LUNGNOT20 2353452 92 CCAGCTACCG AAGCACTGGA GAGTGTCATG GAGGCCTACG AGCAGGTCCA AAAGGGACCC 60 CTGAAGCTGA AAGGCGTCGC AGAGCTGGGA GTGACCAAGC GGAAGAAGAA AAAGAAGGA 120 AAAGACAAAG CGAAACTCCT GGAAGCAATG GGAACGAGCA AAAAGAACGA GGAGGAGAA 180 CGGCGCGGCC TGGACAAGCG GACCCCGGCC CAGGCGGCCT TCGAGAAAAT GCAGGAGAA 240 CGGCAAATGG AAAGGATCCT AAAGAAGGCA TCCAAAACCC ACAAGCAGAG AGTGGAGGA 300 TTCAACAGAC ACCTGGACAC ACTCACGGAG CATTACGACA TTCCCAAAGT CAGCTGGAC 360 AAGTAGCCGC CTGCCCCCAG TATGGAGCAG CATCGAGGGT TCGCAAAAGG CCACACTGG 420 GTTGTGTGTG TTTCCTTTGG TATATTCTGG AAACATGGCT ACACACACCC TTGCATCTT 480 TGCTACAGAC TGCTTTTCGA AGCTGTGTAC CCTCATTCTG GAACTTGATT AAAGTAAGA 540 CGTCCTTGTA CTCAGTTTAG GCTTCTTGGC AACATACAGA AGATACACCC TTTTCGTTT 600 GATGGAAAGT TTCTAAGTTT ATCCAGAGGT AAAGCCCATT GTGTGTCTGT GTCATGTAA 659 1572 base pairs nucleic acid single linear THP1NOT03 2469611 93 GGAAGGGGAA GTTTCGCCTC AGAAGGCTGC CTCGCTGGTC CGAATTCGGT GGCGCCACGT 60 CCGCCCGTCT CCGCCTTCTG CATCGCGGCT TCGGCGGCTT CCACCTAGAC ACCTAACAG 120 CGCGGAGCCG GCCGCGTCGT GAGGGGGTCG GCACGGGGAG TCGGGCGGTC TTGTGCATC 180 TGGCTACCTG TGGGTCGAAG ATGTCGGACA TCGGAGACTG GTTCAGGAGC ATCCCGGCG 240 TCACGCGCTA TTGGTTCGCC GCCACCGTCG CCGTGCCCTT GGTCGGCAAA CTCGGCCTC 300 TCAGCCCGGC CTACCTCTTC CTCTGGCCCG AAGCCTTCCT TTATCGCTTT CAGATTTGG 360 GGCCAATCAC TGCCACCTTT TATTTCCCTG TGGGTCCAGG AACTGGATTT CTTTATTTG 420 TCAATTTATA TTTCTTATAT CAGTATTCTA CGCGACTTGA AACAGGAGCT TTTGATGGG 480 GGCCAGCAGA CTATTTATTC ATGCTCCTCT TTAACTGGAT TTGCATCGTG ATTACTGGC 540 TAGCAATGGA TATGCAGTTG CTGATGATTC CTCTGATCAT GTCAGTACTT TATGTCTGG 600 CCCAGCTGAA CAGAGACATG ATTGTATCAT TTTGGTTTGG AACACGATTT AAGGCCTGC 660 ATTTACCCTG GGTTATCCTT GGATTCAACT ATATCATCGG AGGCTCGGTA ATCAATGAG 720 TTATTGGAAA TCTGGTTGGA CATCTTTATT TTTTCCTAAT GTTCAGATAC CCAATGGAC 780 TGGGAGGAAG AAATTTTCTA TCCACACCTC AGTTTTTGTA CCGCTGGCTG CCCAGTAGG 840 GAGGAGGAGT ATCAGGATTT GGTGTGCCCC CTGCTAGCAT GAGGCGAGCT GCTGATCAG 900 ATGGCGGAGG CGGGAGACAC AACTGGGGCC AGGGCTTTCG ACTTGGAGAC CAGTGAAGG 960 GCGGCCTCGG GCAGCCGCTC CTCTCAAGCC ACATTTCCTC CCAGTGCTGG GTGCGCTT 1020 CAACTGCGTT CTGGCTAACA CTGTTGGACC TGACCCACAC TGAATGTAGT CTTTCAGT 1080 GAGACAAAGT TTCTTAAATC CCGAAGAAAA ATATAAGTGT TCCACAAGTT TCACGATT 1140 CATTCAAGTC CTTACTGCTG TGAAGAACAA ATACCAACTG TGCAAATTGC AAAACTGA 1200 ACATTTTTTG GTGTCTTCTC TTCTCCCCTT TCCGTCTGAA TAATGGGTTT TAGCGGGT 1260 TAGTCTGCTG GCATTGAGCT GGGGCTGGGT CACCAAACCC TTCCCAAAAG GACCCTTA 1320 TCTTCTCTTG CACACATGCC TCTCTCCCAC TTTTCCCAAC CCCCACATTT GCAACTAG 1380 GAGGTTTGCC ATAAAATTGC TCTGCCCTTG ACAAGTTCTG TTAATTTATT GACTTTTG 1440 AAGGCCTGGT CACAACAATC ATATTTCACG TATTTTCCCC CTTTGGTGGC ANGACTGT 1500 GCAATAGGGG GAGAAGACAA GCAGCGGATG GAAGCGTTTT TCTCAAGTTT TGGGAATT 1560 TTCGANCTGA CA 1572 3520 base pairs nucleic acid single linear LIVRTUT04 2515476 94 GAGAAGCCAA GGAAGGAAAC AGGGAAAAAT GTCGCCATGA AGGCCGAGAA CCGCTGCCGC 60 CGCCGACCCC CGCCGGCCCT GAACGCCATG AGCCTGGGTC CCCGCCGCGC CCGCTCCGC 120 TCGACTGCCG TCGCCGCCGA GGCCCCCGTT GATGCCGCTG AGCTCCCCCA ACGCCGCCG 180 CACCGCCTCC GACATGGACA AGAACAGCGG CTCCAACAGC TCCTCCGCCT CTTCGGGCA 240 CAGCAAAGGG CAACAGCCGC CCCGCTCCGC CTCGGCGGGG CCAGCCGGCG AGTCTAAAC 300 CAAGAGCGAT GGAAAGAACT CCAGTGGATC CAAGCGTTAT AATCGCAAAC GTGAACTTT 360 CTACCCCAAA AATGAAAGTT TTAACAACCA GTCCCGTCGC TCCAGTTCAC AGAAAAGCA 420 GACTTTTAAC AAGATGCCTC CTCAAAGGGG CGGCGGCAGC AGCAAACTCT TTAGCTCTT 480 TTTTAATGGT GGAAGACGAG ATGAGGTAGC AGAGGCTCAA CGGGCAGAGT TTAGCCCTG 540 CCAGTTCTCT GGTCCTAAGA AGATCAACCT GAACCACTTG TTGAATTTCA CTTTTGAAC 600 CCGTGGCCAG ACGGGTCACT TTGAAGGCAG TGGACATGGT AGCTGGGGAA AGAGGAACA 660 GTGGGGACAT AAGCCTTTTA ACAAGGAACT CTTTTTACAG GCCAACTGCC AATTTGTGG 720 GTCTGAAGAC CAAGACTACA CAGCTCATTT TGCTGATCCT GATACATTAG TTAACTGGG 780 CTTTGTGGAA CAAGTGCGCA TTTGTAGCCA TGAAGTGCCA TCTTGCCCAA TATGCCTCT 840 TCCACCTACT GCAGCCAAGA TAACCCGTTG TGGACACATC TTCTGCTGGG CATGCATCC 900 GCACTATCTT TCACTGAGTG AGAAGACGTG GAGTAAATGT CCCATCTGTT ACAGTTCTG 960 GCATAAGAAG GATCTCAAGA GTGTTGTTGC CACAGAGTCA CATCAGTATG TTGTTGGT 1020 TACCATTACG ATGCAGCTGA TGAAGAGGGA GAAAGGGGTG TTGGTGGCTT TGCCCAAA 1080 CAAATGGATG AATGTAGACC ATCCCATTCA TCTAGGAGAT GAACAGCACA GCCAGTAC 1140 CAAGTTGCTG CTGGCCTCTA AGGAGCAGGT GCTGCACCGG GTAGTTCTGG AGGAGAAA 1200 AGCACTAGAG CAGCAGCTGG CAGAGGAGAA GCACACTCCC GAGTCCTGCT TTATTGAG 1260 AGCTATCCAG GAGCTCAAGA CTCGGGAAGA GGCTCTGTCG GGATTGGCCG GAAGCAGA 1320 GGAGGTCACT GGTGTTGTGG CTGCTCTGGA ACAACTGGTG CTGATGGCTC CCTTGGCG 1380 GGAGTCTGTT TTTCAACCCA GGAAGGGTGT GCTGGAGTAT CTGTCTGCCT TCGATGAA 1440 AACCACGGAA GTTTGTTCTC TGGACACTCC TTCTAGACCT CTTGCTCTCC CTCTGGTA 1500 AGAGGAGGAA GCAGTGTCTG AACCAGAGCC TGAGGGGTTG CCAGAGGCCT GTGATGAC 1560 GGAGTTAGCA GATGACAATC TTAAAGAGGG GACCATTTGC ACTGAGTCCA GCCAGCAG 1620 ACCCATCACC AAGTCAGGCT TCACACGCCT CAGCAGCTCT CCTTGTTACT ACTTTTAC 1680 AGCGGAAGAT GGACAGCATA TGTTCCTGCA CCCTGTGAAT GTGCGCTGCC TCGTGCGG 1740 GTACGGCAGC CTGGAGAGGA GCCCCGAGAA GATCTCAGCA ACTGTGGTGG AGATTGCT 1800 CTACTCCATG TCTGAGGATG TTCGACAGCG TCACAGATAT CTCTCTCACT TGCCACTC 1860 CTGTGAGTTC AGCATCTGTG AACTGGCTTT GCAACCTCCT GTGGTCTCTA AGGAAACC 1920 AGAGATGTTC TCAGATGACA TTGAGAAGAG GAAACGTCAG CGCCAAAAGA AGGCTCGG 1980 GGAACGCCGC CGAGAGCGCA GGATTGAGAT AGAGGAGAAC AAGAAACAGG GCAAGTAC 2040 AGAAGTCCAC ATTCCCCTCG AGAATCTACA GCAGTTTCCT GCCTTCAATT CTTATACC 2100 CTCCTCTGAT TCTGCTTTGG GTCCCACCAG CACCGAGGGC CATGGGGCCC TCTCCATT 2160 TCCTCTCAGC AGAAGTCCAG GTTCCCATGC AGACTTTCTG CTGACCCCTC TGTCACCC 2220 TGCCAGTCAG GGCAGTCCCT CATTCTGCGT TGGGAGTCTG GAAGAAGACT CTCCCTTC 2280 TTCCTTTGCC CAGATGCTGA GGGTTGGAAA AGCAAAAGCA GATGTGTGGC CCAAAACT 2340 TCCAAAGAAA GATGAGAACA GCTTAGTTCC TCCTGCCCCT GTGGACAGCG ACGGGGAG 2400 TGATAATTCA GACCGTGTTC CTGTGCCCAG TTTTCAAAAT TCCTTCAGCC AAGCTATT 2460 AGCAGCCTTC ATGAAACTGG ACACACCAGC TACTTCAGAT CCCCTCTCTG AAGAGAAA 2520 AGGAAAGAAA AGAAAAAAAC AGAAACAGAA GCTCCTGTTC AGCACCTCAG TCGTCCAC 2580 CAAGTGACAC TACTGGCCCA GGCTACCTTC TCCATCTGGT TTTTGTTTTT GTTTTTTT 2640 CCCCCATGCT TTTGTTTGGC TGCTGTAATT TTTAAGTATT TGAGTTTGAA CAGATTAG 2700 CTGGGGGGAG GGGGTTTCCA CAATGTGAGG GGGAACCAAG AAAATTTTAA ATACAGTG 2760 TTTTCCAGCT TCCTGTCTTT ACACCAAAAT AAAGTATTGA CACAAGAGAT CTCTTCCT 2820 CAAGGTTTTT AGTTCATTGC CAGTTTAGTC TTTTTGACCC ATGTGTAATT AATTTTTC 2880 AACCCAAAGT AAGATTGAGT CCCCTTTGAG ATGCATTAGA GCAGTCCAAC CCAGAATG 2940 ACACACTGCT CTGCTGTACC ATCATGTCAG GGCTTCCTGG ACTCAGTACA CCTCTCAG 3000 TGTCTTTTAA AAAACAGCTG AATCTTTACT ACCTATTTAG TTCTCCTTGT TAAAGAAA 3060 GGGGTGGGAA TAAAATGGAT TTAGGACACC CAGTTTGAAT TGCAGTTTTT TTTTTTCT 3120 CACATGGCCA GGCTGTGGTG CCAGCTTAAT GGAGTAGGCT GTCCTTGGCA CTTGCATG 3180 TGAAAGGAGG GTTTTGCCTC TTCTTGAGCA TGGCTTGAGT TGGTAAGGAA AGCTGTAA 3240 CACGAAGCCC TGAGACCTGC TACCCCTAAG ATCGAGCTTG TTTTCAGTGA CTGGCTTG 3300 TCATAGGAGG AGGAGTCTGG TACAGCTGCA GGAGAGCAGG GCCATCTGAA GCGGTAGC 3360 TGCCACCATC TCCCTCTCAT CTAGAGCAGT TTTCTTATGC CTTGGTTTGA GCTGAATT 3420 ATGTGAATTC TTTTGCTGCT TAATAAAGTG ACCTCTAGGT GCATTAGAAT GCGAAGGC 3480 ATAGTTGCAA TAAATCACCT GCACAAGCAA AAAAAAAAAA 3520 1904 base pairs nucleic acid single linear THP1AZS08 2754573 95 CGGGGGAGTC GGAGGAGGTG GCGGCGCTGG ANNTCCTCCC GGGGACCAGC GACCCGGGAG 60 CATGCACGTC GTCGCACCAG CTTCACTGAG GCTTGGAACA GGAACTAATC TCCCTCCAT 120 TCCAACTTGC TTGACAAAGC TCGCTCTTCC TCCAGCCGCT GAGCCGTCCC TTCTCGCCA 180 GTCCCAGAGC AGGCACCGCG CCGAGGCCCC GCCGCTGGAG CGCGAGGACA GTGGGACCT 240 CAGTTTGGGG AAGATGATAA CAGCTAAGCC AGGGAAAACA CCGATTCAGG TATTACACG 300 ATACGGCATG AAGACCAAGA ACATCCCAGT TTATGAATGT GAAAGATCTG ATGTGCAAA 360 ACACGTGCCC ACTTTCACCT TCAGAGTAAC CGTTGGTGAC ATAACCTGCA CAGGTGAAG 420 TACAAGTAAG AAGCTGGCGA AACATAGAGC TGCAGAGGCT GCCATAAACA TTTTGAAAG 480 CAATGCAAGT ATTTGCTTTG CAGTTCCTGA CCCCTTAATG CCTGACCCTT CCAAGCAAC 540 AAAGAACCAG CTTAATCCTA TTGGTTCATT ACAGGAATTG GCTATTCATC ATGGCTGGA 600 ACTTCCTGAA TATACCCTTT CCCAGGAGGG AGGACCTGCT CATAAGAGAG AATATACTA 660 AATTTGCAGG CTAGAGTCAT TTATGGAAAC TGGAAAGGGG GCATCAAAAA AGCAAGCCA 720 AAGGAATGCT GCTGAGAAAT TTCTTGCCAA ATTTAGTAAT ATTTCTCCAG AGAACCACA 780 TTCTTTAACA AATGTAGTAG GACATTCTTT AGGATGTACT TGGCATTCCT TGAGGAATT 840 TCCTGGTGAA AAGATCAACT TACTGAAAAG AAGCCTCCTT AGTATTCCAA ATACAGATT 900 CATCCAGCTG CTTAGTGAAA TTGCCAAGGA ACAAGGTTTT AATATAACAT ATTTGGATA 960 AGATGAACTG AGCGCCAATG GACAATATCA ATGTCTTGCT GAACTGTCCA CCAGCCCC 1020 CACAGTCTGT CATGGCTCCG GTATCTCCTG TGGCAATGCA CAAAGTGATG CAGCTCAC 1080 TGCTTTGCAG TATTTAAAGA TAATAGCAGA AAGAAAGTAA ATCTGGAGCA ACTTAAAA 1140 TCTTTCAGTA GCACATAAAA AGTTCCCCTC TGGCCCCTTC CCAAGTAAAA CTTTTACC 1200 AGTGTTTATG TCTTGTTTCT AAATCTCTTC ATAGATTCCA TCAACACTCC AGATTTAA 1260 ATCTCCTCAT AGTTGTTATT AAGCTCTTTT TAATGGCTTC AACTTTGTAT CAGTATAC 1320 TATTTATAAA CTTTGTACCA CAAGAGAGAG TGTAGCACCC ATTTTACAGT GCCATGCA 1380 TCAGAGAAAG AAACTGCATG TTTGTTGTTG ATGATGAAAT AAAAATGCTA GCGACAGT 1440 TTCTTACTGG TGCTTAAGCT CTTCTTTGCA CAAAGCTTTA TAAAGGGAAT TCAAAGGA 1500 CCCTTTAGAA TTAGAGTCTT GAGGGACAGC ACTAACAGGC CTTTATTAAG TATGATTG 1560 TGTTAAATTT CAGGGAACAT GATTGGTCTG CTGTGTATTT GAATTCATGT AACAAAGA 1620 TGTTACGATG GGATTCTGCT CATTTTATTA AAAAGCTACT GACTTGACTG TCATCCTG 1680 CTTGTTAGCC ATTGTGAATA AGATTTTAAT GTTGATAATT CTGTTATTTA CATATCTC 1740 ATTTACTTTG AAATTCAAAG GTGAAAATAA AAAATGATGG CCTAAGTAAA ATTTACAA 1800 ATACATGTAA TGGGTTACTT CCTTACTTTC TCTAAGCGTA TGCAACCTTG TATTTTTC 1860 TGACATAACC ATACACAGGT GAATTACTAG TTTAAAAAGC ATTT 1904 1621 base pairs nucleic acid single linear TLYMNOT04 2926777 96 ATTTGGGTTT TTAAAGGGAA GGCGTCCGCG CGGCGGCCAT TTTGTCTTGT CGGCTCCTGT 60 GTGTAGGAGG GATTTCGGCC TGAGAGCGGG CCGAGGAGAT TGGCGACGGT GTCGCCCGT 120 CTTTTCGTTG GCGGGTGCCT GGGCTGGTGG GAACAGCCGC CCGAAGGAAG CACCATGAT 180 TCGGCCGCGC AGTTGTTGGA TGAGTTAATG GGCCGGGACC GAAACCTAGC CCCGGACGA 240 AAGCGCACAA ACGTGCGGTG GGACCACGAG AGCGTTTGTA AATATTATCT CTGTGGTTT 300 TGTCCTGCGG AATTGTTCAC AAATACACGT TCTGATCTTG GTCCGTGTGA AAAAATTCA 360 GATGAAAATC TACGAAAACA GTATGAGAAG AGCTCTCGTT TCATGAAAGT TGGCTATGA 420 AGAGATTTTT TGCGATACTT ACAGAGCTTA CTTGCAGAAG TAGAACGTAG GATCAGACG 480 GGCCATGCTC GTTTGGCATT ATCTCAAAAC CAGCAGTCTT CTGGGGCCGC TGGCCCAAC 540 GGCAAAAATG AAGAAAAAAT TCAGGTTCTA ACAGACAAAA TTGATGTACT TCTGCAACA 600 ATTGAAGAAT TAGGGTCTGA AGGAAAAGTA GAAGAAGCCC AGGGGATGAT GAAATTAGT 660 GAGCAATTAA AAGAAGAGAG AGAACTGCTA AGGTCCACAA CGTCGACAAT TGAAAGCTT 720 GCTGCACAAG AAAAACAAAT GGAAGTTTGT GAAGTATGTG GAGCCTTTTT AATAGTAGG 780 GATGCCCAGT CCCGGGTAGA TGACCATTTG ATGGGAAAAC AACACATGGG CTATGCCAA 840 ATTAAAGCTA CTGTAGAAGA ATTAAAAGAA AAGTTAAGGA AAAGAACCGA AGAACCTGA 900 CGTGATGAGC GTCTAAAAAA GGAGAAGCAA GAAAGAGAAG AAAGAGAAAA AGAACGGGA 960 AGAGAAAGGG AAGAAAGAGA AAGGAAAAGA CGAAGGGAAG AGGAAGAAAG AGAAAAAG 1020 AGGGCTCGTG ACAGAGAAAG AAGAAAGAGA AGTCGTTCAC GAAGTAGACA CTCAAGCC 1080 ACATCAGACA GAAGATGCAG CAGGTCTCGG GACCACAAAA GGTCACGAAG TAGAGAAA 1140 AGGCGGACCA GAAGTAGAGA TCGACGAAGA AGCAGAAGCC ATGATCGATC AGAAAGAA 1200 CACAGATCTC GAAGTCGGGA TCGAAGAAGA TCAAAAAGCC GGGATCGAAA GTCATATA 1260 CACAGGAGCA AAAGTCGGGA CAGAGAACAA GATAGAAAAT CCAAGGAGAA AGAAAAGA 1320 GGATCTGATG ATAAAAAAAG TAGTGTGAAG TCCGGTAGTC GAGAAAAGCA GAGTGAAG 1380 ACAAACACTG AATCGAAGGA AAGTGATACT AAGAATGAGG TCAATGGGAC CAGTGAAG 1440 ATTAAATCTG AAGGTGACAC TCAGTCCAAT TAAAACTGAT CTGATAAGAC CTCAGATC 1500 ACAGAGGACT ACTGTTCGAA GATTTTTGGA AGAATACTGA GAACGGCATA AAGTGAAG 1560 CGACATTTAA AAAATGAGGT GAAAGAAAGC TATAGTGGCA TAGAAAAAGT ATAAAGCT 1620 G 1621 1112 base pairs nucleic acid single linear TESTNOT07 3217567 97 CAACGATCGT GGGNCAGGTA GGTGGTTTCT GGTTTGTTGG GGCGTGTGTA TGTGTATTTA 60 GGGGGACTGA AGGGTACGTG GGGCGAAACA AAACCGGCCA TGGCAGCAGC GGAGGAGGA 120 GACGGGGGCC CCGAAGGGCC AAATCGCGAG CGGGGCGGGG CGGGCGCGAC CTTCGAATG 180 AATATATGTT TGGAGACTGC TCGGGAAGCT GTGGTCAGTG TGTGTGGCCA CCTGTACTG 240 TGGCCATGTC TTCATCAGTG GCTGGAGACA CGGCCAGAAC GGCAAGAGTG TCCAGTATG 300 AAAGCTGGGA TCAGCAGAGA GAAGGTTGTC CCGCTTTATG GGCGAGGGAG CCAGAAGCC 360 CAGGATCCCA GATTAAAAAC TCCACCCCGC CCCCAGGGCC AGAGACCAGC TCCGGAGAG 420 AGAGGGGGAT TCCAGCCATT TGGTGATACC GGGGGCTTCC ACTTCTCATT TGGTGTTGG 480 GCTTTTCCCT TTGGCTTTTT CACCACCGTC TTCAATGCCC ATGAGCCTTT CCGCCGGGG 540 ACAGGTGTGG ATCTGGGACA GGGTCACCCA GCCTCCAGCT GGCAGGATTC CCTCTTCCT 600 TTTCTCGCCA TCTTCTTCTT TTTTTGGCTG CTCAGTATTT GAGCTATGTC TGCTTCCTG 660 CCACCTCCAG CCAGAGAAGA ATCAGTATTG AGGGTCCCTG CTGACCCTTC CGTACTCCT 720 GACCCCCTTG ACCCCTCTAT TTCTGTTGGC TAAGGCCAGC CCTGGACATT GTCCAGGAA 780 GCCTGGGGAG GAGGAGTGAA GTCTGTGCAT AGATGGGAGA GCCTTCTGCT CAGAGGCTC 840 CTCAGTAACG TTGTTTAATT CTCTGCCCTG GGGAAGGAGG ATGGATTGAG AGAATGTCT 900 TCTCCTCTCC TAAGTCTTTG CTTTCCCTGA TTTCTTGATT TGATCTTCAA AGGTGGGCA 960 AGTTCCCTCT GACTCTTCCC CCACTCCCCA TCTTACTGAT TTAATTTAAT TTTTCACT 1020 CCAGAGTCTA ATATGGATTC TGACTCTTAA GTGCTTCCGC CCCCTCACTA CCTCCTTT 1080 TACAAATTCA ATAAAAAAGG TGAAATATAA AA 1112 1040 base pairs nucleic acid single linear SPLNNOT10 3339274 98 CGAGCCCNCC CCCAGCGGGA GCTGTGGGGC AGAGGCGCTG CTGTGGTTGG TCAGTCCAGT 60 AAGAAGCCAG CAGGGCTGGG TGCTGGGGCT TCTTCTCCTG AAGGGGCTGC AAGAGGGAA 120 GCTTAGCCAT GTCGTCCTTG ATCAGAAGGG TGATCAGCAC CGCGAAAGCC CCAGGGGCC 180 TTGGACCCTA CAGTCAAGCT GTATTAGTCG ACAGGACCAT TTACATTTCA GGACAGATA 240 GCATGGACCC TTCAAGTGGA CAGCTTGTGT CAGGAGGGGT AGCAGAAGAA GCTAAACAA 300 CTCTTAAAAA CATGGGTGAA ATTCTGAAAG CTGCAGGCTG TGACTTCACT AACGTGGTG 360 AAACAACTGT TCTTCTGGCT GACATAAATG ACTTCAATAC TGTCAATGAA ATCTACAAA 420 AGTATTTCAA GAGTAATTTT CCTGCTAGAG CTGCTTACCA AGTTGCTGCT TTACCCAAA 480 GCAGCCGAAT TGAAATTGAA GCAGTAGCTA TCCAAGGACC ACTGACAACG GCATCACTA 540 AAGTGGGCCC AGTGCTGTGT AGTCTGGAAT TGTTAACATT TTAATTTTTA CAATTGATG 600 AACATCTTAA TTAACCTTTT AATTTTCACA ATTGATGACA GTGTGAGTTT GATGAAAAT 660 TCTGAAGCTA TTATGGAAAT ACCATGTAAT AGGGAGAGTT GAACATGAAT ATTAGAGAA 720 GAATCCAGTT ACTTTTTTAA ATTACACCTG TGTGCACCTG TATTACTGAA TATAGGAAA 780 AGATACCCAT TACATAGTTA CTCAGTAAAC AAAAGAGAAA TACCAGGTAG GAAAGAAGA 840 TTACTATTCC TGAGAAATAA TCAAGAACAT ATTTAATTTA AACTAATGAT GTGAACTAT 900 TAGTTTTGAT GTCCGTTATG TGATTCTGCT TTTACTTGAG TAAAATTAAA GTGTTTAAA 960 TTGAGATCAA GGAGAAGATA GTGGAACAAA ATGTTATATA GATAATATTT TTCTAATG 1020 AATAAAATAG GCAGATTTCC 1040

Claims (20)

What is claimed is:
1. A purified protein comprising an amino acid sequence selected from SEQ ID NOs: 1-49.
2. An isolated polynucleotide comprising a nucleic acid sequence encoding the protein of claim 1 or the complement of the polynucleotide.
3. A composition comprising a polynucleotide of claim 2 and a reporter molecule.
4. An isolated polynucleotide comprising a nucleic acid sequence selected from SEQ ID NOs:50-98 and the complement of the polynucleotide.
5. A vector containing the polynucleotide of claim 2.
6. A host cell containing the vector of claim 5.
7. A method for using a polynucleotide to produce a protein comprising:
a) culturing the host cell of claim 6 under conditions for the expression of the protein, and
b) recovering the protein from the host cell culture.
8. A method for using a polynucleotide to detect expression of a nucleic acid in a sample, the method comprising:
a) hybridizing the polynucleotide of claim 2 to nucleic acids of the sample, thereby forming a hybridization complex, and
b) detecting hybridization complex formation, wherein complex formation indicates the expression of the polynucleotide in the sample.
9. The method of claim 8 wherein the polynucleotide is attached to a substrate or bonded to the surface of a microarray.
10. The method of claim 8 wherein the nucleic acids of the sample are amplified prior to hybridization.
11. A method of using a polynucleotide to screen a plurality of molecules to identify a ligand, the method comprising:
a) combining the polynucleotide of claim 2 with a plurality of molecules under conditions to allow specific binding, and
b) detecting specific binding, thereby identifying a ligand which specifically binds the polynucleotide.
12. The method of claim 11 wherein the molecules are selected from DNA molecules, RNA molecules, peptide nucleic acids, artificial chromosome constructions, peptides, and transcription factors.
13. A method for diagnosing a disease associated with gene expression in a sample containing nucleic acids, the method comprising:
a) hybridizing a polynucleotide of claim 2 to nucleic acids of the sample under conditions to form a hybridization complex,
b) comparing hybridization complex formation with standards, thereby diagnosing the disease.
14. The method of claim 13 wherein expression is diagnostic of cancer or immune response.
15. A composition comprising the protein of claim 1 and a pharmaceutical carrier or a labeling moiety.
16. A method for using a protein to screen a plurality of molecules to identify a ligand, the method comprising:
a) combining the protein of claim 1 with the molecules under conditions to allow specific binding, and
b) detecting specific binding, thereby identifying a ligand which specifically binds the protein.
17. The method of claim 16 wherein the molecules are selected from DNA molecules, RNA molecules, peptide nucleic acids, peptides, pharmaceutical agents, proteins, mimetics, agonists, antagonists, Ned antibodies, immunoglobulins, inhibitors, and drugs.
18. A method of using a protein to prepare and purify antibodies comprising:
a) immunizing a animal with the protein of claim 1 under conditions to elicit an antibody response,
b) isolating animal antibodies,
c) attaching the protein to a substrate,
d) contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein,
e) dissociating the antibodies from the protein, thereby obtaining purified antibodies.
19. An antibody which specifically binds a protein of claim 1.
20. A method for using an antibody to detect protein expression in a sample, the method comprising:
a) combining the antibody of claim 19 with a sample under conditions to form antibody:protein complexes, and
b) detecting complex formation with standards, wherein detection indicates expression of the protein in the sample.
US09/840,787 1997-09-23 2001-09-26 Human regulatory molecules Abandoned US20020058264A1 (en)

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US09/877,633 US20020102569A1 (en) 1997-09-23 2001-06-08 Diagnostic marker for cancers
US09/840,787 US20020058264A1 (en) 1997-09-23 2001-09-26 Human regulatory molecules
US10/247,451 US20040018188A9 (en) 1999-01-20 2002-09-18 Sparc-related proteins

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US08/933,750 US5932442A (en) 1997-09-23 1997-09-23 Human regulatory molecules
US09/234,613 US6132973A (en) 1997-09-23 1999-01-20 Human regulatory molecules
US51886500A 2000-03-03 2000-03-03
US09/840,787 US20020058264A1 (en) 1997-09-23 2001-09-26 Human regulatory molecules

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US09/349,015 Continuation-In-Part US20020015950A1 (en) 1999-01-20 1999-07-07 Atherosclerosis-associated genes
US09/877,633 Continuation-In-Part US20020102569A1 (en) 1997-09-23 2001-06-08 Diagnostic marker for cancers

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US08/965,689 Expired - Fee Related US6015702A (en) 1997-09-23 1997-11-06 Human ubiquitin-conjugating enzymes
US09/234,613 Expired - Fee Related US6132973A (en) 1997-09-23 1999-01-20 Human regulatory molecules
US09/877,633 Abandoned US20020102569A1 (en) 1997-09-23 2001-06-08 Diagnostic marker for cancers
US09/840,787 Abandoned US20020058264A1 (en) 1997-09-23 2001-09-26 Human regulatory molecules

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US08/965,689 Expired - Fee Related US6015702A (en) 1997-09-23 1997-11-06 Human ubiquitin-conjugating enzymes
US09/234,613 Expired - Fee Related US6132973A (en) 1997-09-23 1999-01-20 Human regulatory molecules
US09/877,633 Abandoned US20020102569A1 (en) 1997-09-23 2001-06-08 Diagnostic marker for cancers

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AU9403798A (en) 1999-04-12
WO1999015658A3 (en) 1999-06-10
US5932442A (en) 1999-08-03
CA2304485A1 (en) 1999-04-01
US20020102569A1 (en) 2002-08-01
WO1999015658A2 (en) 1999-04-01
US6015702A (en) 2000-01-18
EP1017809A2 (en) 2000-07-12

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