US20040058358A1

US20040058358A1 - Lipid metabolism transcription factor

Info

Publication number: US20040058358A1
Application number: US10/443,200
Authority: US
Inventors: Henry Yue; Matthew Kaser; Mariah Baughn
Original assignee: Incyte Corp
Current assignee: Incyte Corp
Priority date: 1999-05-26
Filing date: 2003-05-21
Publication date: 2004-03-25
Also published as: CA2374376A1; AU5018900A; JP2003501026A; WO2000073443A1; US6245526B1; EP1181364A1

Abstract

The invention provides a mammalian nucleic acid and fragments thereof. It also provides for the use of these nucleic acids in a model system for the characterization, diagnosis, evaluation, treatment of conditions, diseases and disorders associated with expression of the mammalian nucleic acid. The invention additionally provides expression vectors and host cells for the production of the protein encoded by the mammalian nucleic acid.

Description

This application is a contiunation of U.S. Ser. No. 09/709,976, filed Nov. 10, 2000, which is a divisional of U.S. Ser. No. 09/318,978 filed May 26, 1999.[0001]

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of a new mammalian protein and to the use of these sequences in the characterization, diagnosis, and treatment of cell proliferative and lipid disorders.

BACKGROUND OF THE INVENTION

Phylogenetic relationships among organisms have been demonstrated many times, and studies from a diversity of prokaryotic and eukaryotic organisms suggest a more or less gradual evolution of biochemical and physiological mechanisms and metabolic pathways. Despite different evolutionary pressures, proteins that regulate the cell cycle in yeast, nematode, fly, rat, and man have common chemical or structural features and modulate the same general cellular activity. Comparisons of human gene sequences with those from other organisms where the structure and/or function may be known allow researchers to draw analogies and to develop model systems for testing hypotheses. These model systems are of great importance in developing and testing diagnostic and therapeutic agents for human conditions, diseases and disorders.

Fatty acids are required for phospholipid, glycolipid, hormone, and intracellular messenger formation; to anchor proteins to membranes; and as fuel molecules. Most cells can synthesize fatty acids from acetate substrates, though many mammalian cells also obtain fatty acids by hydrolysis of triglycerides. Synthesis of phospholipids primarily occurs on the surface of the smooth endoplasmic reticulum. Although most cells constitutively form fatty acids, the level of synthesis varies with the needs of the cell. During phases of rapid cell division, membrane formation requires enhanced production of phospholipids. Animals that have fasted and are then fed high-carbohydrate, low-fat diets show marked increases in the amount and activity of enzymes responsible for fatty acid synthesis. Increased synthesis of long chain fatty acids also occurs in multiple common neoplasms, including those arising in the breast, prostate, ovary, colon, and endometrium. Overexpression of fatty acid synthase (FAS), a major enzyme of fatty acid biosynthesis, is a marker for poor prognosis in breast tumors and has been shown to be important for tumor growth (Moncur et al. (1997) Proc Natl Acad Sci 95:6989-6994).

The transcriptional regulation of enzymes involved in fatty acid synthesis is associated with Spot 14 (S14) protein. S14 is a small, acidic nuclear protein with a carboxyterminal “zipper” domain involved in homodimer formation. It is expressed in tissues that produce lipids for use as metabolic fuels, such as lactating mammary tissue, white and brown adipose tissue, and liver. The expression of S14 is increased in response to insulin, dietary carbohydrates, glucose, and thyroid hormone and reduced in response to glucagon, fasting, and in diabetes mellitus. Expression of antisense oligonucleotides has shown S14 induces tissue-specific expression of several lipogenic enzymes including FAS and ATP citrate lyase. The S14 gene is located on chromosome 11 at position q13.5, a chromosomal region amplified in approximately 20% of breast cancers, and is expressed in several breast cancer-derived cell lines and in a majority of primary breast tumors (Cunningham et al. (1998) Thyroid 8:815-825; Liaw and Towle (1984) J Biol Chem 259:7253-7260; Brown et al. (1997) J Biol Chem 272:2163-2166; and Moncur, supra).

A zebrafish gastrulation protein, G12, shares features with S14 including acidic pI (˜4.9) and nearly identical size (˜17 kDa). The sequence similarity between the two proteins is strongest at the carboxyterminus, including the zipper domain. G12 is expressed in an outer, enveloping layer of cells (EVL), analogous to the mammalian trophectoderm, during a period in gastrulation in which the EVL layer expands to cover the developing, embryonic deep cell layer. During this stage, apical membrane turnover in the EVL increases and raises the requirement for phospholipids used in plasma membranes (Conway (1995) Mech Dev 52:383-391; Fink and Cooper (1996) Dev Biol 174:180-189).

The discovery of a polynucleotide encoding a new mammalian protein satisfies a need in the art by providing new compositions which are useful in the characterization, diagnosis, and treatment of cell proliferative and lipid disorders.

SUMMARY OF THE INVENTION

The invention is based on the discovery of a polynucleotide encoding a mammalian protein, lipid metabolism transcription factor (LMTF), which satisfies a need in the art by providing new compositions useful in the characterization, diagnosis, and treatment of cell proliferative and lipid disorders.

The invention provides an isolated and purified mammalian polynucleotide comprising the nucleic acid sequence of SEQ ID NO:1 or a fragment thereof. The invention also provides fragments homologous to the mammalian polynucleotide from rat, mouse, and monkey.

The invention further provides an isolated and purified polynucleotide or a fragment thereof which hybridizes under high stringency conditions to the polynucleotide of SEQ ID NO:1. The invention also provides an isolated and purified polynucleotide which is complementary to the polynucleotide of SEQ ID NO:1. In one aspect, a single stranded complementary RNA or DNA molecule is used as a probe which hybridizes under high stringency conditions to the mammalian polynucleotide or a fragment thereof.

The invention further provides a method for detecting a polynucleotide in a sample containing nucleic acids, the method comprising the steps of: (a) hybridizing a probe to at least one of the nucleic acids of the sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide in the sample. In one aspect, the method further comprises amplifying the polynucleotide prior to hybridization. The polynucleotide or fragment thereof may comprise an element or target on a microarray. The invention also provides a method for screening a library of molecules for specific binding to a polynucleotide or a fragment thereof, the method comprising providing a library of molecules, combining the polynucleotide of

claim

1 with a plurality of molecules under conditions which allow specific binding, and detecting binding of the polynucleotide to each of a plurality of molecules, thereby identifying at least one molecule which specifically binds the polynucleotide. Such molecules are potential regulators of polynucleotide function.

The invention also provides an expression vector containing at least a fragment of the polynucleotide of SEQ ID NO:1. In another aspect, the expression vector is contained within a host cell. The invention further provides a method for producing a protein, the method comprising the steps of culturing the host cell for expression of the protein and recovering the protein from the host cell culture. The invention also provides an isolated and purified protein comprising the amino acid sequence of SEQ ID NO:2 or a portion thereof. Additionally, the invention provides a composition comprising a purified protein having the sequence of SEQ ID NO:2 or a portion thereof in conjunction with a pharmaceutical carrier.

The invention further provides a method for using a portion of the protein to produce antibodies. The invention also provides a method for using a protein or a portion thereof to screen for molecules which specifically bind the protein, the method comprising the steps of combining the protein or a portion thereof with a library of molecules under conditions which allow complex formation and detecting complex formation, wherein the presence of the complex identifies a molecule which specifically binds the protein. In one aspect, a molecule identified using the method increases the activity of the protein. In another aspect, a molecule identified using the method decreases the activity of the protein.

BRIEF DESCRIPTION OF THE FIGURES AND TABLE

FIGS. 1A, 1B, [0014] 1C, 1D, 1E, and 1F show the nucleic acid sequence (SEQ ID NO:1) encoding the amino acid sequence (SEQ ID NO:2) of the mammalian protein. The alignment was produced using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco Calif.).
FIGS. 2A and 2B show the chemical and structural similarity between SEQ ID NO:2, G12 ([0015] GI 861207; SEQ ID NO:26) and Spot14 (GI 1171574; SEQ ID NO:27), produced using the multisequence alignment program of LASERGENE software (DNASTAR, Madison Wis.). The amino acids of SEQ ID NO:2, from residue 45 to residue 59, may be used for antibody production.
Table 1 shows the ESTs from human, rat, mouse, and monkey which have homology with SEQ ID NO:1 and includes their nucleotide length, biological source, region of overlap with SEQ ID NO:1, and percent identity with SEQ ID NO:1.[0016]

DESCRIPTION OF THE INVENTION

It is understood that this invention is not limited to the particular machines, materials and methods described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims. As used herein, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. For example, a reference to “a host cell” includes a plurality of such host cells known to those skilled in the art. [0017]
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commnonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors 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. [0018]
Definitions [0019]
“LMTF” refers to a purified protein, lipid metabolism transcription factor, obtained from any mammalian species, including murine, bovine, ovine, porcine, simian, and preferably the human species, and from any source, whether natural, synthetic, semi-synthetic, or recombinant. [0020]
“Agents, molecules, or compounds” are used interchangably and refer to that which interacts with, specifically binds to, or modifies the expression of the polynucleotides and proteins of the invention; and may be composed of at least one of the following: nucleic acids, proteins, carbohydrates, fats, lipids, organic and inorganic substances. [0021]
“Biologically active” refers to a protein having structural, immunological, regulatory, or chemical functions of a naturally occurring, recombinant or synthetic molecule. [0022]
“Complementary” refer to the natural base pairing by hydrogen bonding between purines and pyrimidines. For example, the sequence A-C-G-T forms hydrogen bonds with its complement T-G-C-A or U-G-C-A. Two single-stranded molecules may be considered partially complementary, if only some of the nucleotides bond, or completely complementary, if nearly all of the nucleotides bond. The degree of complementarity between nucleic acid strands affects the efficiency and strength of the hybridization and amplification reactions. [0023]
“Derivative” refers to the chemical modification of a polynucleotide or protein sequence. Chemical modifications of a sequence can include replacement of hydrogen by an alkyl, acyl, or amino group or glycosylation, pegylation, or any similar process which retains or enhances biological activity or lifespan of the molecule. [0024]
“Fragment” refers to an Incyte clone or any part of a polynucleotide which retains a usable, functional characteristic. Useful fragments include oligonucleotides which may be used in hybridization or amplification technologies or in regulation of replication, transcription or translation. [0025]
“Hybridization complex” refers to a complex between two nucleic acid sequences by virtue of the formation of hydrogen bonds between purines and pyrimidines. [0026]
“Polynucleotide” refers to a nucleic acid molecule, oligonucleotide, or any fragment thereof. It may be DNA or RNA of genomic or synthetic origin, double-stranded or single-stranded, and combined with carbohydrate, lipids, protein or other materials to perform a particular activity such as transformation or form a useful composition such as a peptide nucleic acid (PNA). “Oligonucleotide” is equivalent to the terms amplimer, primer, oligomer, element, target, and probe and is preferably single stranded. [0027]
“Protein” refers to an oligopeptide, peptide, or polypeptide or portions thereof whether naturally occurring or synthetic. [0028]
“Portion”, as used herein, refers to any part of a protein used for any purpose, but especially for the screening of molecules or compounds which specifically bind to that part or for the production of antibodies. [0029]
“Sample” is used in its broadest sense. A sample containing nucleic acids may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue; a tissue print; and the like. [0030]
Molecules or compounds which “specifically bind” the mammalian polynucleotide or protein may include, nucleic acids, carbohydrates, lipids, proteins, or any other organic or inorganic molecules or their combinations which stabilize, increase, or decrease the activity of the mammalian polynucleotide or protein. “Purified” refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably about 75% free, and most preferably about 90% free, from other components with which they are naturally associated. [0031]
“Substrate” refers to any rigid or semi-rigid support to which polynucleotides or proteins are bound and includes membranes, filters, chips, sides, wafers, fibers, magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores. [0032]

THE INVENTION

The invention is based on the discovery of a new mammalian polynucleotide which encodes a mammalian protein, lipid metabolism transcription factor, and the use of the nucleic acid sequence, or fragments thereof, and amino acid sequences, or portions thereof, as compositions in the characterization, diagnosis, or treatment, of cell proliferative and lipid disorders. [0033]
Nucleic acids encoding the mammalian protein of the present invention were identified by BLAST using Incyte clone 700145292H1 which was differentially expressed in male rat reproductive tissue. A consensus sequence, SEQ ID NO:1, was assembled from the following overlapping and/or extended nucleic acid fragments found in Incyte Clones 1479946F6, 3241390F6, 1432520R1, 4534217H1, 2191992H1, 1320132T1, 1516707T1, 5595953H1, and 1988906R6; SEQ ID NOs:3-11, respectively. FIGS. [0034] 1A-1F show the concensus sequence and translation of SEQ ID NO:1.
In one embodiment, the protein comprising the amino acid sequence of SEQ ID NO:2, LMTF, is 183 amino acids in length and has one potential N-glycosylation site at residue N77; three potential protein kinase C phosphorylation sites at residues S96, T162, and T169; and a potential leucine zipper motif from residue L154 through L168. As shown in FIGS. 2A and 2B, the protein has chemical and structural similarity with zebrafish G12 ([0035] GI 861207; SEQ ID NO:26) and mouse S14 (GI 1171574; SEQ ID NO:27). In particular, LMTF shares 48% identity with G12 protein and 32% identity with S14. LMTF, G12, and S14 are similar in size (20 kDa, 17.5 kDa, and 17 kDa, respectively) and isoelectric point (5.3, 5.0, and 4.8, respectively), as calculated using LASERGENE software (DNASTAR). Furthermore, LMTF, G12, and S14 share conserved leucine residues comprising a zipper motif at residues L154, L161, and L168 in LMTF.
Table 1 shows the nucleic acid fragments from human, rat, mouse, and monkey and their sequence coverage and identity with SEQ ID NO:1. [0036] Columns 1 and 2 list the SEQ ID NO and Incyte clone number, respectively, for each nucleic acid fragment. The fragments of SEQ ID NO:1, SEQ ID NOs:3-11, are useful in hybridization or amplification technologies to identify and distinguish between the mammalian molecules disclosed herein and similar sequences including SEQ ID NOs:12-25. Column 3 lists the nucleotide length for each fragment. Columns 4 and 5 identify the source organism and Incyte cDNA library from which the fragments were isolated, respectively. Column 6 identifies the range of nucleotide residues in SEQ ID NO:1 over which each fragment shows identity. Column 7 shows the percent sequence identity between each fragment and SEQ ID NO:1 over the nucleotides set forth in column 6.
Northern analysis shows the expression of LMTF in various libraries, particularly in nervous tissues of human, rat, and monkey. Of particular note is the expression of LMTF in conditions associated with cell proliferation, such as cancer and inflammation. [0037]
The mammalian fragments comprising SEQ ID NO:12-13 from monkey, SEQ ID NO:14-15 from mouse, and SEQ ID NO:16-25 from rat were identified using either SEQ ID NO:1 or SEQ ID NOs:3-11. These fragments may be used to obtain the full length sequence for a particular species which in turn can be used to produce transgenic animals which mimic human diseases. The fragments are useful in hybridization and amplication technologies to monitor animal toxicological studies, clinical trials, and subject/patient treatment profiles through time. [0038]
Characterization and Use of the Invention [0039]
In a particular embodiment disclosed herein, mRNA was isolated from mammalian cells and tissues using methods which are well known to those skilled in the art and used to prepare the cDNA libraries. The Incyte clones listed above were isolated from mammalian cDNA libraries. At least one library preparation representative of the invention is described in the EXAMPLES below. The consensus mammalian sequence was chemically and/or electronically assembled from fragments including Incyte clones, extension, and/or shotgun sequences using computer programs such as the AUTOASSEMBLER application (Applied Biosystems, Foster City Calif.). [0040]
Methods for sequencing nucleic acids are well known 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, T7 SEQUENASE DNA polymerase, Taq DNA polymerase, and THERMOSEQUENASE DNA polymerase (Amersham Pharmacia Biotech (APB), Picataway N.J.) or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Rockville Md.). Preferably, sequence preparation is automated with machines such as the HYDRA microdispenser (Robbins Scientific, Sunnyvale Calif.), MICROLAB 2200 system (Hamilton, Reno Nev.), and the DNA ENGINE thermal cycler (MJ Research, Watertown Mass.). Machines used for sequencing include the ABI 3700, 377 or 373 DNA sequencing systems (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (APB), and the like. The sequences may be analyzed using a variety of algorithms which are well known in the art and described in Ausubel (1997; [0041] Short Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., unit 7.7) and in Meyers (1995; Molecular Biology and Biotechnology, Wiley VCH, New York N.Y., pp. 856-853).
Shotgun sequencing is used to generate more sequence from cloned inserts derived from multiple sources. Shotgun sequencing methods are well known in the art and use thermostable DNA polymerases, heat-labile DNA polymerases, and primers chosen from representative of regions flanking the nucleic acid sequences of interest. Prefinished sequences (incomplete assembled sequences) are inspected for identity using various algorithms or programs well known in the art (Gordon (1998) Genome Res 8:195-202). Contaminating sequences including vector or chimeric sequences or deleted sequences can be removed or restored, respectively, organizing the prefinished sequences into finished sequences. [0042]
The sequences of the invention may be extended using various PCR-based methods known in the art. For example, the XL-PCR kit (Applied Biosystems), nested primers, and commercially available EDNA or genomic DNA libraries (Life Technologies; Clontech, Palo Alto Calif., respectively) may be used to extend the nucleotide sequence. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO software (Molecular Biology Insights, Cascade Colo.) to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to a target sequence at temperatures of about 68° C. to 72° C. When extending a sequence to recover regulatory elements, it is preferable to use genomic, rather than cDNA libraries. [0043]
The polynucleotide sequence of SEQ ID NO:1 and fragments thereof can be used in various hybridization technologies for various purposes. Hybridization probes may be designed or derived from SEQ ID NO:1. Such probes maybe made from a highly specific region such as the 5′ regulatory region or from a conserved motif, and used in protocols to identify naturally occurring sequences encoding the mammalian protein, allelic variants, or related sequences, and should preferably have at least 50% sequence identity to any of the protein sequences. The hybridization probes of the subject invention may be DNA or RNA and maybe derived from the sequence of SEQ ID NO:1 or from genomic sequences including promoters, enhancers, and introns of the mammalian gene. Hybridization or PCR probes may be produced using oligolabeling, nick translation, end-labeling, or PCR amplification in the presence of the labeled nucleotide. A vector containing the nucleic acid sequence may be used to produce an mRNA probe in vitro by addition of an RNA polymerase and labeled nucleotides. These procedures may be conducted using commercially available kits such as those provided by APB. [0044]
The stringency of hybridization is determined by G+C content of the probe, salt concentration, and temperature. In particular, stringency can be increased by reducing the concentration of salt or raising the hybridization temperature. In solutions used for some membrane based hybridizations, additions of an organic solvent such as formamide allows the reaction to occur at a lower temperature. Hybridization can be performed at low stringency with buffers, such as 5×SSC with 1% sodium dodecyl sulfate (SDS) at 60° C., which permits the formation of a hybridization complex between nucleotide sequences that contain some mismatches. Subsequent washes are performed at higher stringency with buffers such as 0.2×SSC with 0.1% SDS at either 45° C. (medium stringency) or 68° C. (high stringency). At high stringency, hybridization complexes will remain stable only where the nucleic acid sequences are completely complementary. In some membrane-based hybridizations, preferably 35% or most preferably 50%, formamide can be added to the hybridization solution to reduce the temperature at which hybridization is performed, and background signals call be reduced by the use of other detergents such as Sarkosyl or TRITON X-100 (Sigma-Aldrich, St. Louis Mo.) and a blocking agent such as salmon sperm DNA. Selection of components and conditions for hybridization are well known to those skilled in the art and are reviewed in Ausubel (supra) and Sambrook et al. (1989) [0045] Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.
Microarrays may be prepared and analyzed using methods known in the art. Oligonucleotides may be used as either probes or targets in a microarray. The microarray can be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and single nucleotide polymorphisms. Such information may be used to determine gene function; to understand the genetic basis of a condition, disease, or disorder; to diagnose a condition, disease, or disorder; and to develop and monitor the activities of therapeutic agents. (See, e.g., Brennan et al. (1995) U.S. Pat. No. 5,474,796; Schena et al. (1996) Proc Natl Acad Sci 93:10614-10619; Baldeschweiler et al. (1995) PCT application WO95/251116; Shalon et al. (1995) PCT application WO95/35505; Heller et al. (1997) Proc Natl Acad Sci 94:2150-2155; and Heller et al. (1997) U.S. Pat. No. 5,605,662.) [0046]
Hybridization probes are also useful in mapping the naturally occurring genomic sequence. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome DNA libraries. [0047]
A multitude of polynucleotide sequences capable of encoding the mammalian protein may be cloned into a vector and used to express the protein, or portions thereof, in host cells. The nucleotide sequence can be engineered by such methods as DNA shuffling (Stenmier and Crameri (1996) U.S. Pat. No. 5,830,721) and site-directed mutagenesis to create new restriction sites, alter glycosylation patterns, change codon preference to increase expression in a particular host, produce splice variants, extend half-life, and the like. The expression vector may contain transcriptional and translational control elements (promoters, enhancers, specific initiation signals, and 3′untranslated regions) from various sources which have been selected for their efficiency in a particular host. The vector, nucleic acid sequence, and regulatory elements are combined using in vitro recombinant DNA techniques, synthetic techniques, and/or in vivo genetic recombination techniques well known in the art and described in Sambrook (supra, ch. 4, 8, 16 and 17). [0048]
A variety of host systems may be transformed with an expression vector. These include, but are not limited to, bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems transformed with baculovirus expression vectors; plant cell systems transformed with expression vectors containing viral and/or bacterial elements, or animal cell systems (Ausubel supra, unit 16). For example, an adenovirus transcription/translation complex may be utilized in mammalian cells, Sequences may be ligated into the non-essential E1 or E3 region of the viral genome, and the infective virus used to transform and express the protein in host cells. The Rous sarcoma virus enhancer or SV40 or EBV-based vectors may also be used for high-level protein expression [0049]
Routine cloning, subcloning, and propagation of polynucleotide sequences can be achieved using the multifunctional PBLUESCRIPT vector (Stratagene, La Jolla Calif.) or PSPORT1 plasmid (Life Technologies). Introduction of a polynucleotide sequence into the multiple cloning site of these vectors disrupts the lacZ gene and allows colorimetric screening for transformed bacteria. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. [0050]
For long term production of recombinant proteins, the vector can be stably transformed into cell lines along with a selectable or visible marker gene on the same or on a separate vector. After transformation, cells are allowed to grow for about 1 to 2 days in enriched media and then are transferred to selective media. Selectable markers, antimetabolite, antibiotic, or herbicide resistance genes, confer resistance to the relevant selective agent and allow growth and recovery of cells which successfully express the introduced sequences. Resistant clones identified either by survival on selective media or by the expression of visible markers, such as anthocyanins, green fluorescent protein (GFP), β glucuronidase, luciferase and the like, may be propagated using tissue culture techniques. Visible markers are also used to quantify the amount of protein expressed by the introduced genes. Verification that the host cell contains the desired mammalian polynucleotide is based on DNA-DNA or DNA-RNA hybridizations or PCR amplification techniques. [0051]
The host cell may be chosen for its ability to modify a recombinant protein in a desired fashion. Such modifications include acetylation, carboxylation, glycosylation, phosphorylation, lipidation, acylation and the like. Post-translational processing which cleaves a “prepro” form may also be used to specify protein targeting, folding, and/or activity. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (CHO, HEK293, and WVI38; American Type Culture Collection, Manassas Va.) may be chosen to ensure the correct modification and processing of the foreign protein. [0052]
Heterologous moieties engineered into a vector for ease of purification include glutathione S-transferase (GST), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and the like. GST, CBP, and 6-His are purified using commercially available affinity matrices such as immobilized glutathione, calmodulin, and metal-chelate resins, respectively. FLAG and c-myc are purified using commercially available monoclonal and polyclonal antibodies. A proteolytic cleavage site may be located between the desired protein sequence and the heterologous moiety for ease of separation following purification. Methods for recombinant protein expression and purification are discussed in Ausubel (supra, unit 16) and are commercially available. [0053]
Proteins or portions thereof may be produced not only by recombinant methods, but also by using chemical methods well known in the art. Solid phase peptide synthesis may be carried out in a batchwise or continuous flow process which sequentially adds α-amino- and side chain-protected amino acid residues to an insoluble polymeric support via a linker group. A linker group such as methylamine-derivatized polyethylene glycol is attached to poly(styrene-co-divinylbenzene) to form the support resin. The amino acid residues are N-α-protected by acid labile Boc (t-butyloxycarbonyl) or base-labile Fmoc (9-fluorenylmethoxycarbonyl). The carboxyl group of the protected amino acid is coupled to the amine of the linker group to anchor the residue to the solid phase support resin. Trifluorloacetic acid or piperidine are used to remove the protecting group in the case of Boc or Fmoc, respectively. Each additional amino acid is added to the anchored residue using a coupling agent or pre-activated amino acid derivative, and the resin is washed. The full length peptide is synthesized by sequential deprotection, coupling of derivitized amino acids, and washing with dichloromethane and/or N,N-dimethylformamide. The peptide is cleaved between the peptide carboxyterminus and the linker group to yield a peptide acid or amide (Novabiochem 1997/98 Catalog and Peptide Synthesis Handbook, San Diego Calif. pp. S1-S20). Automated synthesis may also be carried out on machines such as the ABI 431A peptide synthesizer (Applied Biosystems). A protein or portion thereof may be purified by preparative high performance liquid chromatography and its composition confirmed by amino acid analysis or by sequencing (Creighton (1984) [0054] Proteins, Structures and Molecular Properties, WH Freeman, New York N.Y.).
Various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with mammalian protein or any portion thereof. Adjuvants such as Freund's, mineral gels, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemacyanlin (KLH), and dinitroplhenol may be used to increase immunological response. The oligopeptide, peptide, or portion of protein used to induce antibodies should consist of at least about five amino acids, more preferably ten amino acids, which are identical to a portion of the natural protein. Oligopeptides may be fused with proteins such as KLH in order to produce antibodies to the chimeric molecule. [0055]
Monoclonal antibodies may be prepared using any technique which provides for the production of antibodies 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. (See, e.g., Kohler et al. (1975) Nature 256:495-497; Kozbor et al. (1985) J Immunol Methods 81:31-42; Cote et al. (1983) Proc Natl Acad Sci 80:2026-2030; and Cole et al. (1984) Mol Cell Biol 62:109-120.) [0056]
Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce epitope-specific single chain antibodies. Antibody fragments which contain specific binding sites for epitopes of the mammalian protein may also be generated. For example, such fragments include, but are not limited to, F(ab′)2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide 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. (See, e.g., Huse et al. (1989) Science 246:1275-1281.) [0057]
The mammalian protein may be used in screening assays of phagemid or B-lymphocyte immunoglobulin libraries to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoassays 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 the protein and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes is preferred, but a competitive binding assay may also be employed (Pound (1998) [0058] Immunochemical Protocols, Humana Press, Totowa N.J.).
A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid, amino acid, and antibody assays. Synthesis of labeled molecules may be achieved using Promega (Madison Wis.) or APB kits for incorporation of a labeled nucleotide such as [0059] ³²P-dCTP, Cy3-dCTP or Cy5-dCTP or amino acid such as ³⁵S-methionine (APB). Nucleic acids and amino acids may be directly labeled with a variety of substances including fluorescent, chemiluminescent, or chromogenic agents, and the like, by chemical conjugation to amines, thiols and other groups present in the molecules using reagents such as BIODIPY or FITC (Molecular Probes, Eugene Oreg.).
Diagnostics [0060]
The polynucleotides, fragments, oligonucleotides, complementary RNA and DNA molecules, and PNAs may be used to detect and quantify altered gene expression, absence/presence vs. excess, expression of mRNAs or to monitor mRNA levels during therapeutic intervention. Condition, diseases or disorders associated with altered expression of LMTF include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease, myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; and a lipid disorder such as fatty liver, cholestasis, carnitine deficiency, carnitine palmitoyltransferase deficiency, myoadenylate deaminase deficiency, hypertriglyceridemia, lipid storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy, adrenoleukodystrophy, GM[0061] ₂gangliosidosis, and ceroid lipofuscinosis, abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, diabetes mellitus, lipodystrophy, lipomatoses, acute panniculitis, disseminated fat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimal change disease, lipomas, hypercholesterolemia, hypercholesterolemia with hypertriglyceridemia, primary hypoalphalipoproteinemia, hypothyroidism, renal disease, liver disease, lecithin:cholesterol acyltransferase deficiency, cerebrotendinous xanthomatosis, sitosterolemia, hypocholesterolemia, Tay-Sachs disease, Sandhoff's disease, hyperlipidemia, hyperlipemia, lipid myopathies, and obesity. The diagnostic assay may use hybridization or amplification technology to compare gene expression in a biological sample from a patient to standard samples in order to detect altered gene expression. Qualitative or quantitative methods for this comparison are well known in the art.
For example, the nucleotide sequence may be labeled by standard methods and added to a biological sample from a patient. After an incubation period in which hybridization complexes form, the sample is washed and the amount of label, or its signal, is quantified and compared with a standard value. If the amount of label in the patient sample is significantly altered in comparison to the standard value, then the presence of the associated condition, disease or disorder is indicated. [0062]
In order to provide a basis for the diagnosis of a condition, disease or disorder associated with gene expression, a normal or standard expression profile is established. This may be accomplished by combining a biological sample taken from normal subjects, either animal or human, with a sequence or a fragment thereof under conditions for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of an isolated and purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a particular condition, disease, or disorder. Deviation from standard values toward those associated with a particular condition is used to diagnose that condition. [0063]
Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies and in clinical trial or to monitor the treatment of an individual patient. Once the presence of a condition is established and a treatment protocol is initiated, diagnostic assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in a normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months. [0064]
Therapeutics [0065]
Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of the mammalian LMTF, zebrafish G12, and mouse Spot14. In addition, expression is closely associated with nervous tissue and appears to play a role in cell proliferative and inflammatory disorders. In the treatment of conditions associated with increased expression or activity, it is desirable to decrease expression or protein activity. In the treatment of conditions associated with decreased expression or activity, it is desirable to increase expression or protein activity. [0066]
In one embodiment, the mammalian protein or a portion or derivative thereof may be administered to a subject to treat or prevent a condition associated with altered expression or activity of the mammalian protein. Examples of such conditions include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease, myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; and a lipid disorder such as fatty liver, cholestasis, carnitine deficiency, carnitine palmitoyltransferase deficiency, myoadenylate deaminase deficiency, hypertriglyceridemia, lipid storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy, adrenoleukodystrophy, GM[0067] ₂gangliosidosis, and ceroid lipofuscinosis, abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, diabetes mellitus, lipodystrophy, lipomatoses, acute panniculitis, disseminated fat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimal change disease, lipomas, hypercholesterolemia, hypercholesterolemia with hypertriglyceridemia, primary hypoalphalipoproteinemia, hypothyroidism, renal disease, liver disease, lecithin:cholesterol acyltransferase deficiency, cerebrotendinous xanthomatosis, sitosterolemia, hypocholesterolemia, Tay-Sachs disease, Sandhoff's disease, hyperlipidemia, hyperlipemia, lipid myopathies, and obesity.
In another embodiment, a composition comprising the purified mammalian protein in conjunction with a pharmaceutical carrier may be administered to a subject to treat or prevent a condition associated with altered expression or activity of the mammalian protein including, but not limited to, those provided above. [0068]
In a further embodiment, an agonist which modulates the activity of the mammalian protein may be administered to a subject to treat or prevent a condition associated with altered expression or activity of the protein including, but not limited to, those listed above. [0069]
In an additional embodiment, a vector capable of expressing the mammalian protein or a portion or derivative thereof may be administered to a subject to treat or prevent a condition associated with altered expression or activity of protein including, but not limited to, those described above. [0070]
In yet another embodiment, an antagonist or inhibitor of the mammalian protein may be administered to a subject to treat or prevent a condition associated with altered expression or activity of the protein. In one aspect, an antibody which specifically binds the mammalian protein 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 the mammalian protein. [0071]
In a still further embodiment, a vector expressing the complement of the polynucleotide encoding the mammalian protein may be administered to a subject to treat or prevent a condition associated with altered expression or activity of the protein including, but not limited to, those described above. [0072]
Any of the nucleic acids, complementary sequences, vectors, proteins, agonists, antagonists, or antibodies 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. A combination of therapeutic agents may act synergistically to effect treatment of a particular condition at a lower dosage of each agent. [0073]
Gene expression may be modified by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5′, or regulatory regions of the gene encoding the mammalian protein. Oligonucleotides designed with reference to the transcription initiation site are preferred. Similarly, inhibition can be achieved using triple helix base-pairing which inhibits the binding of polymerases, transcription factors, or regulatory molecules (Gee et al. In: Huber and Carr (1994) [0074] Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco N.Y., pp. 163-177.) A complementary sequence may also be designed to block translation by preventing binding between ribosomes and mRNA.
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 at sites such as GUA, GUU, and GUC. Once such sites are identified, an oligonucleotide with the same sequence may be evaluated for secondary structural features which would render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing their hybridization with complementary oligonucleotides using ribonuclease protection assays. [0075]
Complementary nucleic acids and ribozymes of the invention may be prepared via recombinant expression, in vitro or in vivo, or using solid phase phosphoramidite chemical synthesis. In addition, RNA molecules may be modified to increase intracellular stability and half-life by addition of flanking sequences at the 5′ and/or 3′ ends of the molecule or by the use of phosphorothioate or 2′ O-ethyl rather than phosphodiesterase linkages within the backbone of the molecule. Modification is inherent in the production of PNAs and can be extended to other nucleic acid molecules. Either the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, and or the modification of adenine, cytidine, guanine, thymine, and uridine with acetyl-, methyl-, thio- groups renders the molecule less available to endogenous endonucleases. [0076]
The nucleic acid sequence encoding the mammalian protein may be used to screen a library of molecules for specific binding affinity. The assay can be used to screen a library of DNA molecules, RNA molecules, PNAs, peptides, or proteins including transcription factors, enhancers, repressors, and the like which regulate the activity of the nucleic acid sequence in the biological system. The assay involves providing a library of molecules, combining the mammalian nucleic acid sequence or a fragment thereof with the library of molecules under conditions to allow specific binding, and detecting specific binding to identify at least one molecule which specifically binds the nucleic acid sequence. [0077]
Similarly the mammalian protein or a portion thereof may be used to screen libraries of molecules in any of a variety of screening assays. The portion of the protein employed in such screening may be free in solution, affixed to an abiotic or biotic substrate (e.g. borne on a cell surface), or located intracellularly. Specific binding between the protein and molecule may be measured. Depending oil the kind of library being screened, the assay may be used to identify DNA, RNA, or PNA molecules, agonists, antagonists, antibodies, immunoglobulins, inhibitors, peptides, proteins, drugs and the like, which specifically bind the protein. One method for high throughput screening using very small assay volumes and very small amounts of test compound is described in U.S. Pat. No. 5,876,946, which screens large numbers of molecules for enzyme inhibition or receptor binding. [0078]
Pharmaceutical compositions are those substances wherein the active ingredients are contained in an effective amount to achieve a desired and intended purpose. The determination of an effective dose is well within the capability of those skilled in the art. For any compound, the therapeutically effective dose may be estimated initially either in cell culture assays or in animal models. The animal model is also used to achieve a desirable concentration range and route of administration. Such information may then be used to determine useful doses and routes for administration in humans. [0079]
A therapeutically effective dose refers to that amount of protein or inhibitor which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity of such agents 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 toxic and therapeutic effects is the therapeutic index, and it may be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indexes are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use. [0080]
Model Systems [0081]
Animal models may be used as bioassays where they exhibit a toxic response similar to that of humans and where exposure conditions are relevant to human exposures. Mammals are the most common models and most toxicity studies are performed on rodents such as rats or mice because of low cost, availability, and abundant reference toxicologic. Inbred rodent strains provide a convenient model for investigation of the physiological consequences of under- or over-expression of genes of interest and for the development of methods for diagnosis and treatment of diseases. A rodent strain inbred to over-express a particular gene may also serve as a convenient source of the protein expressed by that gene. [0082]
Toxicology is the study of the effects of agents on living systems. The majority of toxicity studies are performed on rats or mice to help predict the effects of these agents on human health. Observation of qualitative and quantitative changes in physiology, behavior, homeostatic processes, and lethality are used to generate a toxicity profile and to assess the consequences on human health following exposure to the agent. [0083]
Toxicological tests measure the effects of a single, repeated, or long-term exposure of a subject to an agent. Agents may be tested for specific endpoints such as cytotoxicity, mutagenicity, carcinogenicity and teratogenicity. Degree of response varies according to the route of exposure (contact, ingestion, injection, or inhalation), age, sex, genetic makeup, and health status of the subject. Toxicokinetic studies trace the absorption, distribution, metabolism, storage, and excretion of the agent in subject tissues, and toxicodynamic studies chart biological responses that are consequences of the presence of the agent in subject tissues. [0084]
Genetic toxicology identifies and analyzes the ability of an agent to produce genetic mutations Genotoxic agents usually have common chemical or physical properties that facilitate interaction with nucleic acids and are most harmful when chromosomal aberrations are passed along to progeny. Toxicological studies may identify agents that increase the frequency of structural or functional abnormalities in progeny if administered to either parent before conception, to the mother during pregnancy, or to the developing organism. Mice and rats are most frequently used in these tests because of their short reproductive cycle and their capacity to be raised in numbers sufficient to satisfy statistical requirements. [0085]
All toxicology studies on experimental animals involve the preparation of a form of the agent for administration, the selection of the route of administration, and the selection of the species to resemble the species of pharmacological interest. Dose concentrations are varied to investigate a range of dose-related effects which are identified, measured, and related to exposure. [0086]
Acute toxicity tests are based on a single administration of the agent to the subject to determine the symptomology or lethality of the agent. Three experiments are conducted: 1) an initial dose-range-finding experiment, 2) an experiment to narrow the range of effective doses, and 3) a final experiment for establishing the dose-response curve. [0087]
Prolonged toxicity tests are based on the repeated administration of the agent. Rat and dog are commonly used in these studies to provide data from species in different families. With the exception of carcinogenesis, there is considerable evidence that daily administration of an agent at high-dose concentrations for periods of three to four months will reveal most forms of toxicity in adult animals. [0088]
Chronic toxicity tests, with a duration of a year or more, are used to demonstrate either the absence of toxicity or the carcinogenic potential of an agent. When studies are conducted on rats, a minimum of three test groups plus one control group are used, and animals are examined and monitored at the outset and at intervals throughout the experiment. [0089]
Transgenic rodents which over-express or under-express a gene of interest may be inbred and used to model human diseases or to test therapeutic or toxic agents. (See, e.g., van Beusechem and Valerio, In: Murray (1992) [0090] Transgenesis: Applications of Gene Transfer, John Wiley & Sons Ltd. Chichester, England, pp. 283-289.) To produce the rat or mouse model, a gene candidate which mimics a human disease is coupled to a strong promoter and injected into a fertilized egg, and the egg transferred into a pseudopregnant dam. The promoter may be activated at a specific time in a specific tissue type during fetal development or postnatally. Expression of the transgene is monitored by analysis of phenotype, tissue-specific mRNA expression, and challenged with experimental drug therapies. Examples of transgenes used as models of human disease include the investigation of the mutant amyloid precursor protein and apolipoprotein E genes in familial Alzheimer's Disease (Price and Sisodia (1998) Ann Rev Neurosci 21:479-505).
Embryonic stem cells (ES) isolated from rodent embryos retain the potential to form an embryo. When ES cells are placed inside a carrier embryo, they resume normal development and contribute to all tissues of the live-born animal. ES cells are the preferred cells used in the creation of experimental knockout and knockin rodent strains. Mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and are grown under culture conditions well known in the art. Vectors for knockout strains contain a disease gene candidate modified to include a marker gene sequence which disrupts transcription and/or translation in vivo. The vector is introduced into ES cells by transformation methods such as electroporation, liposome delivery, microinjection, and the like which are well known in the art. The endogenous rodent gene is replaced by the disrupted disease gene through homologous recombination and integration during cell division. Then transformed ES cells are selected, identified, and preferably microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. [0091]
ES cells are also used to study the differentiation of various cell types and tissues in vitro, such as neural cells, hematopoietic lineages, and cardiomyocytes (Bain et al. (1995) Dev Biol 168:342-357; Wiles and Keller (1991) Development 111:259-267; and Klug et al. (1996) J Clin Invest 98:216-224). Recent developments demonstrate that ES cells derived from human blastocysts may also be manipulated in vitro to differentiate into eight separate cell lineages, including endoderm, mesoderm, and ectodermal cell types (Thomson (1998) Science 282:1145-1147). [0092]
As described herein, the uses of the nucleotide sequences, provided in the Sequence Listing of this application, are exemplary of known techniques and are not intended to reflect any limitation on their use in any technique that would be known to the person of average skill in the art. Furthermore, the nucleotide sequences provided in this application may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known to the person of ordinary skill in the art. [0093]
In gene knockout analysis, a region of a human disease gene candidate is enzymatically modified to include a non-mammalian gene such as the neomycin phosphotransferase gene (neo; Capecchi (1989) Science 244:1288-1292). The inserted coding sequence disrupts transcription and translation of the targeted gene and prevents biochemical synthesis of the disease candidate protein. The modified gene is transformed into cultured embryonic stem cells (described above), the transformed cells are injected into rodent blastulae, and the blastulae are implanted into pseudopregnant dams. Transgenic progeny are crossbred to obtain homozygous inbred lines. [0094]
Totipotent ES cells, present in the early stages of embryonic development, can be used to create knockin humanized animals (pigs) or transgenic animal models (mice or rats) of human diseases. With knockin technology, a region of a human gene is injected into animal ES cells, and the human sequence integrates into the animal cell genome by recombination. Totipotent ES cells which contain the integrated human gene are handled as described above. Inbred animals are studied and treated to obtain information on the analogous human condition. These methods have been used to model several human diseases. (See, e.g., Lee et al. (1998) Proc Natl Acad Sci 95:11371-11376; Baudoin et al. (1998) Genes Dev 12:1202-1216; and Zhuang et al. (1998) Mol Cell Biol 18:3340-3349). [0095]
The field of animal testing deals with data and methodology from basic sciences such as physiology, genetics, chemistry, pharmacology and statistics. These data are paramount in evaluating the effects of therapeutic agents on non-human primates as they can be related to human health. Monkeys are used as human surrogates in vaccine and drug evaluations, and their responses are relevant to human exposures under similar conditions. Cynomolgus monkeys ([0096] Macaca fascicularis, M. mulatta) and common marmosets (Callithrix jacchlus) are the most common non-human primates (NHPs) used in these investigations. Since great cost is associated with developing and maintaining a colony of NHPs, early research and toxicological studies are usually carried out in rodent models. In studies using behavioral measures such as drug addiction, NHPs are the first choice test animal. In addition, NHPs and individual humans exhibit differential sensitivities to many drugs and toxins and can be classified as “extensive metabolizers” and “poor metabolizers” of these agents. For this reason, NHPs are the favored models for studying metabolism and toxicology of agents acted upon by the cytochrome P₄₅₀family of enzymes.
In additional embodiments, the nucleotide sequences which encode the mammalian protein may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions. All patents and publications cited are incorporated herein by reference. [0097]

EXAMPLES

It is to be understood that this invention is not limited to the particular machines, materials and methods described. Although particular embodiments are described, equivalent embodiments may be used to practice the invention. The described embodiments are not intended to limit the scope of the invention which is limited only by the appended claims. The examples below are provided to illustrate the subject invention and are not included for the purpose of limiting the invention. For purposes of example, the preparation of the human corpus callosum cDNA library, CORPNOT02, is described. [0098]
I Representative cDNA Sequence Preparation [0099]
The human corpus callosum cDNA library CORPNOT02 was constructed from tissue obtained from a 74-year-old Caucasian male (specimen #RA95-09-0670; International Institute for the Advancement of Medicine, Exton Pa.) who died from Alzheimer's disease. The frozen tissue was homogenized and lysed in guanidinium isothiocyanate solution using a POLYTRON homogenizer (PT-3000; Brinkmann Instruments, Westbury N.J.). The lysate was centrifuged over a 5.7 M CsCl cushion using an SW28 rotor in an 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 (Life Technologies) at 37° C. The RNA extraction and precipitation were repeated as before. [0100]
Messenger RNA (mRNA) was isolated using the OLIGOTEX kit (Qiagen, Valencia Calif.) and used to construct the cDNA library. The mRNA was handled according to the recommended protocols in the SUPERSCRIPT plasmid system (Life Technologies) which contains a NotI primer-adaptor designed to prime the first strand cDNA synthesis at the poly(A) tail of mRNAs. Double stranded cDNA was blunted, ligated to EcoRI adaptors and digested with NotI (New England Biolabs, Beverly Mass.). The cDNAs were fractionated on a SEPHAROSE CL-4B column (APB), and those cDNAs exceeding 400 bp were ligated into the NotI and EcoRI sites of the pINCY plasmid (Incyte Genomics, Palo Alto Calif.). The plasmid was transformed into DH5α or ELECTROMAX DH10B competent cells (Life Technologies). [0101]
Plasmid DNA was released from the cells and purified using the REAL PREP 96 plasmid kit (Qiagen). 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 inoculation, the cultures were incubated for 19 hours and then lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid DNA 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 at 4° C. [0102]
The cDNAs were prepared using either a MICROLAB 2200 system (Hamilton) or a HYDRA microdispenser (Robbins Scientific) in combination with the DNA ENGINE thermal cyclers (MJ Research) and sequenced by the method of Sanger and Coulson (1975; J Mol Biol 94:441-448) using either ABI PRISM 377 (Applied Biosystems) or MEGABACE 1000 (APB) sequencing, systems. Most of the isolates were sequenced according to standard ABI protocols and kits (Applied Biosytems). The solution volumes were used at 0.25x-1.0x concentrations. In the alternative, cDNAs were sequenced using solutions and dyes from APB. [0103]
II Identification, Extension, Assembly, and Analyses of the Sequences [0104]
Incyte clone 700145292 from ZOOSEQ database (Incyte Genomics) was used to identify [0105] Incyte Clone 5595953 from the LIFESEQ database (Incyte Genomics). The first pass and extended cDNAs, SEQ ID NOs:3-11, which cluster with Incyte Clone 5595953 were assembled using Phred/Phrap or CONSED (Green, University of Washington, Seattle Wash.) or the GCG Fragment assembly system (Genetics Computer Group (GCG), Madison Wis.). The assembled sequence was searched for open reading frames, and the coding region was translated using MACDNASIS PRO software (Hitachi Software Engineering). The full length nucleotide and amino acid sequences were analyzed by BLAST queries against databases such as the GenBank databases, SwissProt, BLOCKS, PRINTS, Prosite, and PFAM and by LASERGENE software (DNASTAR). Functional analyses of the amino acid sequences were performed using MOTIFS (GCG) and HMM algorithms. Antigenic index (Jameson-Wolf analysis) of the amino acid sequences were determined using LASERGENE software (DNASTAR). Then, the clones and assembled sequence were compared using BLAST across all mammalian libraries to identify homologous nucleic acid sequences, SEQ ID NOs:12-25.
III Sequence Similarity [0106]
Sequence similarity was calculated as percent identity based on comparisons between at least two nucleic acid or amino acid sequences using the clustal method of the MEGALIGN program (DNASTAR). The clustal method uses an algorithm which groups sequences into clusters by examining the distances between all pairs. After the clusters are aligned pairwise, they are realigned in groups. Percent similarity between two sequences, sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of very low or zero similarity between the two sequences are not included. [0107]
IV Northern Analysis [0108]
Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. [0109]
Analogous computer techniques applying BLAST were used to search for identical or related molecules in nucleotide databases such as GenBank or LIFESEQ (Incyte Genomics). Sequence-based analysis is much faster than membrane-based hybridization, and the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score which is defined as: (percent sequence identity×percent mammalian BLAST score) divided by 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% to 2% error, and with a product score of at least 70, the match will be exact. Similar or related molecules are usually identified by selecting those which show product scores between 8 and 40. [0110]
The results of northern analyses are reported is a percentage distribution of libraries in which the transcript encoding the mammalian protein occurred. Analysis involved the categorization of cDNA libraries by organ/tissue and disease. The organ/tissue categories included cardiovascular, dermatologic, developmental, endocrine, gastrointestinal, hematopoietic/immune, musculoskeletal, nervous, reproductive, and urologic. The disease categories included cancer, inflammation/trauma, cell proliferation, and neurological. For each category, the number of libraries expressing the sequence was counted and divided by the total number of libraries across all categories. [0111]
V Extension of Polynucleotides [0112]
The nucleic acid sequence of SEQ ID NO:1 was produced by extension of Incyte cDNA clones using oligonucleotide primers. One primer was synthesized to initiate 5′ extension of the known fragment, and the other, to initiate 3′ extension of the known fragment. The initial primers were designed using OLIGO software (Molecular Biology Insights) to be about 22 to 30 nucleotides in length, to have a GC content of about 50%, and to anneal to the target sequence at temperatures of about 68° C. to about 72° C. Any fragment which would result in hairpin structures and primer-primer dimerizations was avoided. Selected human cDNA libraries were used to extend the sequence. If more than one extension is needed, additional or nested sets of primers are designed. [0113]
High fidelity amplification was obtained by performing PCR in 96-well plates using the DNA ENGINE thermal cycler (MJ Research). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg[0114] ²⁺, (NH₄)₂SO₄, and β-mercaptoethanol, TAQ DNA polymerase (APB), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair selected from the plasmid: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1 min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min; Step 7: storage at 4° C. In the alternative, parameters for the primer pair, T7 and SK+(Stratagene), were as follows: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 57° C., 1 min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min; and Step 7: storage at 4° C.
The concentration of DNA in each well was determined by dispensing 100 μl PICOGREEN quantitation reagent (0.25% (v/v); Molecular Probes) dissolved in 1× TE and 0.5 μl of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Life Sciences, Acton Mass.) and allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 μl to 10 μl aliquot of the reaction mixture was analyzed by electrophoresis on a 1% agarose mini-gel to determine which reactions were successful in producing longer sequence. [0115]
The extended sequences were desalted, concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison Wis.), and sonicated or sheared prior to religation into pUC 18 vector (APB). For shotgun sequencing, the digested fragments were separated on about 0.6-0.8% agarose gels, fragments were excised as visualized under UV light, and agar removed/digested with AGARACE (Promega). Extended fragments were religated using T4 ligase (New England Biolabs, Beverly Mass.) into pUC 18 vector (APB), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transformed into competent [0116] E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37° C. in 384-well plates in LB/2× carbenicillin liquid media.
The cells were lysed, and DNA was amplified using Taq DNA polymerase (APB) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1 min; Step 4: 72° C., 2 min; Step 5: [0117] steps 2, 3, and 4 repeated 29 times; Step 6: 72° C., 5 min; and Step 7: storage at 4° C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the conditions described above. Samples were diluted with 20% dimethysulphoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (APB) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, the nucleotide sequence of SEQ ID NO:1 is used to obtain regulatory sequences using the procedure above, oligonucleotides designed for outward extension, and a genomic library. [0118]
VI Labeling of Probes anti Hybridization Analyses [0119]
Polynucleotide sequences are isolated from a biological source and applied to a substrate for standard nucleic acid hybridization protocols by one of the following methods. A mixture of target nucleic acids, a restriction digest of genomic DNA, is fractionated by electrophoresis through all 0.7% agarose gel in 1× TAE [Tris-acetate-ethylenediamine tetraacetic acid (EDTA)] running buffer and transferred to a nylon membrane by capillary transfer using 20× saline sodium citrate (SSC). Alternatively, the target nucleic acids are individually ligated to a vector and inserted into bacterial host cells to form a library. Target nucleic acids are arranged on a substrate by one of the following methods. In the first method, bacterial cells containing individual clones are robotically picked and arranged on a nylon membrane. The membrane is placed on bacterial growth medium, LB agar containing carbenicillin, and incubated at 37° C. for 16 hours. Bacterial colonies are denatured, neutralized, and digested with proteinase K. Nylon membranes are exposed to UV irradiation in a STRATALINKER UV-crosslinker (Stratagene) to cross-link DNA to the membrane. [0120]
In the second method, target nucleic acids are amplified from bacterial vectors by thirty cycles of PCR using primers complementary to vector sequences flanking the insert. Amplified target nucleic acids are purified using SEPHACRYL-400 beads (APB). Purified target nucleic acids are robotically arrayed onto a glass microscope slide (Corning Life Sciences). The slide was previously coated with 0.05% aminopropyl silane (Sigma-Aldrich) and cured at 110° C. The arrayed glass slide (microarray) is exposed to UV irradiation in a STRATALINKER UV-crosslinker (Stratagene). [0121]
cDNA probe sequences are made from mRNA templates. Five micrograms of mRNA is mixed with 1 μg random primer (Life Technologies), incubated at 70° C. for 10 minutes, and lyophilized. The lyophilized sample is resuspended in 50 μl of 1× first strand buffer (cDNA Synthesis systems; Life Technologies) containing a dNTP mix, [α-[0122] ³²P]dCTP, dithiothreitol, and MMLV reverse transcriptase (Stratagene), and incubated at 42° C. for 1-2 hours. After incubation, the probe is diluted with 42 μl dH₂O, heated to 95° C. for 3 minutes, and cooled on ice. mRNA in the probe is removed by alkaline degradation. The probe is neutralized, and degraded mRNA and unincorporated nucleotides are removed using a PROBEQUANT G-50 MicroColumn (APB). Probes can be labeled with fluorescent nucleotides, Cy3-dCTP or Cy5-dCTP (APB), in place of the radiolabeled nucleotide, [³²P]dCTP.
Hybridization is carried out at 65° C. in a hybridization buffer containing 0.5 M sodium phosphate (pH 7.2), 7% SDS, and 1 mM EDTA. After the substrate is incubated in hybridization buffer at 65° C. for at least 2 hours, the buffer is replaced with 10 ml of fresh buffer containing the probe sequences. After incubation at 65° C. for 18 hours, the hybridization buffer is removed, and the substrate is washed sequentially under increasingly stringent conditions, up to 40 mM sodium phosphate, 1% SDS, 1 mM EDTA at 65° C. To detect signal produced by a radiolabeled probe hybridized on a membrane, the substrate is exposed to a PHOSPHORIMAGER cassette (APB), and the image is analyzed using IMAGEQUANT data analysis software (APB). To detect signals produced by a fluorescent probe hybridized on a microarray, the substrate is examined by confocal laser microscopy, and images are collected and analyzed using GEMTOOLS gene expression analysis software (Incyte Genomics). [0123]
VII Complementary Polynucleotides [0124]
Sequences complementary to the polynucleotide, or a fragment thereof, are used to detect, decrease, or inhibit gene expression. Although use of oligonucleotides comprising from about 15 to about 30 base pairs is described, essentially the same procedure is used with larger or smaller fragments or their derivatives (PNAs). Oligonucleotides are designed using OLIGO software (Molecular Biology Insights) and SEQ ID NO:1 or its fragments, SEQ ID NO:3-9. To inhibit transcription by preventing promoter binding, a complementary oligonucleotide is designed to bind to the most unique 5′ sequence, most preferably about 10 nucleotides before the initiation codon of the open reading frame. To inhibit translation, a complementarily oligonucleotide is designed to prevent ribosomal binding to the mRNA encoding the mammalian protein. [0125]
VIII Expression of the Mammalian Protein [0126]
Expression and purification of the mammalian protein are achieved using bacterial or virus-based expression systems. For expression in bacteria, cDNA is subcloned into a vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into bacterial hosts, e.g., BL21(DE3). Antibiotic resistant bacteria express the mammalian protein upon induction with isopropyl beta-D-thiogalactopyranoside. Expression in eukaryotic cells is achieved by infecting [0127] Spodoptera frugiperda (Sf9) insect cells with recombinant baculovirus, Autographica californica nuclear polyhedrosis virus. The nonessential polyhedrin gene of baculovirus is replaced with the mammalian cDNA by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
In most expression systems, the mammalian protein is synthesized as a fusion protein with GST or FLAG, which permits rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates. GST enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (APB). Following purification, the GST moiety can be proteolytically cleaved from the mammalian protein at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak, Rochester N.Y.). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (Qiagen). Methods for protein expression and purification are discussed in Ausubel (supra, unit 16). Purified mammalian protein obtained by these methods can be used directly in the following activity assay. [0128]
IX Functional Assays [0129]
Protein function is assessed by expressing the sequences encoding LMTF at physiologically elevated levels in mammalian cell culture. The polynucleotide is subcloned into pCMV SPORT vector (Life Technologies), which contains the strong cytomegalovirus promoter, and 5-10 μg of the vector is transformed into a endothelial or hematopoietic human cell line using electroporation. An additional 1-2 μg of a plasmid containing sequence encoding CD64-GFP (Clontech) is co-transformed to provide an fluorescent marker to identify transformed cells using flow cytometry. [0130]
The influence of the introduced genes on expression can be assessed using purified populations of these transformed cells. Since CD64-GFP, which is expressed on the surface of transformed cells, binds to conserved regions of human immunoglobulin G (IgG), the transformed cells is separated using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success N.Y.). mRNA is purified from the cells and analyzed by hybridization techniques. [0131]
X Production of LMTF Specific Antibodies [0132]
LMTF is purified using polyacrylamide gel electrophoresis is used to immunize rabbits and to produce antibodies using standard protocols. [0133]
Alternatively, the amino acid sequence of LMTF is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity. An immunogenic epitope such as those near the C-terminus or in hydrophilic regions is selected, synthesized, and used to raise antibodies by means known to those of skill in the art. [0134]
Typically, epitopes of about 15 residues in length are produced using an ABI 431A peptide synthesizer (Applied Biosystems) using Fmoc-chemistry and coupled to KLH (Sigma-Aldrich) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester to increase immunogenicity. Rabbits are immunized with the epitope-KLH complex in complete Freund's adjuvant. Immunizations are repeated at intervals thereafter in incomplete Freund's adjuvant. After a sufficient period of time, antisera are drawn and tested for antipeptide activity. Testing involves binding the peptide to plastic, blocking with 1% bovine serum albumin, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG. Methods well known in the art are used to determine antibody titer and the amount of complex formation. [0135]
XI Purification of Naturally Occurring Protein Using Specific Antibodies [0136]
Naturally occurring or recombinant mammalian protein is purified by immunoaffinty chromatography using antibodies specific for the protein. An immunoaffinity column is constructed by covalently coupling the antibody to CNBr-activated SEPHAROSE resin (APB). Media containing the protein is passed over the immunoaffinity column, and the column is washed using high ionic strength buffers in the presence of detergent to allow preferential absorbance of the protein. After coupling, the column is eluted using a buffer of pH 2-3 or a high concentration of urea or thiocyanate ion to disrupt antibody/protein binding, and the protein is collected. [0137]
XII Screening Molecules for Specific Binding with the Polynucleotide or Protein [0138]
The nucleic acid sequence, or fragments thereof, or the protein, or portions thereof, are labeled with [0139] ³²P-dCTP, Cy3-dCTP, Cy5-dCTP (APB), or BIODIPY or FITC (Molecular Probes), respectively. Libraries of candidate molecules previously arranged on a substrate are incubated in the presence of labeled nucleic acid sequence or protein. After incubation, the substrate is washed, and any position on the substrate retailing label, which indicates specific binding or complex formation, is assayed, and the binding molecule is identified. Data obtained using different concentrations of the nucleic acid or protein are used to calculate affinity between the labeled nucleic acid or protein and the bound molecule.
XIII Demonstration of Protein Activity [0140]
LMTF activity is measured by its ability to modulate transcription of a reporter gene. The assay entails the use of a reporter gene construct that consists of a transcription factor response element fused upstream to sequences encoding the [0141] E. coli β-galactosidase enzyme (LacZ). Sequences encoding LMTF are subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression. Vectors of choice include PCMV SPORT (Life Technologies) and PCR 3.1 (Invitrogen, Carlsbad Calif.), both of which contain the cytomegalovirus promoter. The recombinant vector and reporter gene construct are co-transformed into a human cell line, preferably of neuronal origin, using either liposome formulations or electroporation. The amount of β-galactosidase enzyme activity associated with LMTF transfectcd cells, relative to control cells transformed with the reporter construct alone, is proportional to the amount of transcription modulated by the LMTF gene product.

All patents and publications mentioned in the specification are incorporated by reference herein. 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 that are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.

TABLE 1


Nucleic Acid	Incyte	Nucleotide				Percent
SEQ ID NO:	Clone Number	Length	Source	Library	Coverage	Identity

3	1479946F6	502	Homo sapiens	CORPNOT02	1-502	n/a
4	3241390F6	529	Homo sapiens	COLAUCT01	281-801	n/a
5	1432520R1	562	Homo sapiens	BEPINON01	599-1178	n/a
6	4534217H1	254	Homo sapiens	OVARNOT12	1152-1414	n/a
7	2191992H1	238	Homo sapiens	THYRTUT03	1200-1437	n/a
8	1320132T1	661	Homo sapiens	BLADNOT04	1299-1957	n/a
9	1516707T1	624	Homo sapiens	PANCTUT01	1445-2070	n/a
10	5595953H1	252	Homo sapiens	COLCDTT03	1592-1845	n/a
11	1988906R6	302	Homo sapiens	LUNGAST01	1851-2092	n/a
12	700712962H1	144	Macaca fascicularis	MNBFNOTO2	12-156	92.4
13	700715135H1	274	Macaca fascicularis	NBCNOT01	292-564	93.1
14	701253541H1	273	Mus musculus	MOLUDIT07	560-1032	71.2
15	701252210H1	250	Mus musculus	MOLUDIT07	1564-1831	81.0
16	700545683H1	272	Rattus norvegicus	RASPNOT01	1-271	52.2
17	700145292H1	257	Rattus norvegicus	RAPRNOT01	191-488	44.7
18	700861443H1	239	Rattus norvegicus	RABGNOT02	338-591	63.2
19	700225363H1	302	Rattus norvegicus	RAKINOT01	479-780	86.4
20	700643425H1	286	Rattus norvegicus	RABUNOT01	593-879	81.1
21	700525920H1	285	Rattus norvegicus	RABMNOT01	783-1066	77.2
22	700773927H1	270	Rattus norvegicus	RABONOT01	1001-1197	54.8
23	700513679H1	283	Rattus norvegicus	RASNNOT01	1318-1594	72.1
24	700767486H1	266	Rattus norvegicus	RAHYNOT01	1594-1876	73.7
25	700327166H1	199	Rattus norvegicus	RASNNOT01	1813-2008	74.4

[0143]

0

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 27

<210> SEQ ID NO 1

<211> LENGTH: 2092

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 5595953

<400> SEQUENCE: 1

gggcctttta tctcggtgct gccgggggag gcgggaggag gagacaccag gggtggccct 60

gagcgccggc gacacctttc ctggactata aattgagcac ctgggatggg tagggggcca 120

acgcagtcac cgccgtccgc agtcacagtc cagccactga ccgcagcagc gcccttgcgt 180

acagccgctt gcagcgagaa cactgaattg ccaacgagca ggagagtctc aaggcgcaag 240

aggaggccag ggctcgaccc acagagcacc ctcagccatc gcgagtttcc gggcgccaaa 300

gccaggagaa gccgcccatc ccgcagggcc ggtctgccag cgagacgaga gttggcgagg 360

gcggaggagt gccgggaatc ccgccacacc ggctatagcc aggcccccag cgcgggcctt 420

ggagagcgcg tgaaggcggg catccccttg acccggccga ccatccccgt gcccctgcgt 480

ccctgcgctc caacgtccgc gcggccacca tgatgcaaat ctgcgacacc tacaaccaga 540

agcactcgct ctttaacgcc atgaatcgct tcattggcgc cgtgaacaac atggaccaga 600

cggtgatggt gcccagcttg ctgcgcgacg tgcccctggc tgaccccggg ttagacaacg 660

atgttggcgt ggaggtaggc ggcagtggcg gctgcctgga ggagcgcacg cccccagtcc 720

ccgactcggg aagcgccaat ggcagctttt tcgcgccctc tcgggacatg tacagccact 780

acgtgcttct caagtccatc cgcaacgaca tcgagtgggg ggtcctgcac cagccgcctc 840

caccggctgg gagcgaggag ggcagtgcct ggaagtccaa ggacatcctg gtggacctgg 900

gccacttgga gggtgcggac gccggcgaag aagacctgga acagcagttc cactaccacc 960

tgcgcgggct gcacactgtg ctctcgaaac tcacgcgcaa agccaacatc ctcactaaca 1020

gatacaagca ggagatcggc ttcggcaatt ggggccactg aggcgtggcg cccgtggctg 1080

cccagcacct tcttcgaccc atctcaccct ctctcattcc tcaaagcttt tttttttttt 1140

cctggctggg gggcgggaag ggcagactgc aaactggggg gctgcgtacg tgcaggaggc 1200

gcggtggggc tgcgtggagg agggggccac gtgtgagaga gaagaaaatg gtggccggag 1260

atgggagggc ccaaggaacc tcctgggagg gggcctgcat tctatgttgg tgggaatggg 1320

actgggctga cgccctgcat tcagcctgtg cctttcctgg ggtttctttt ctgttctttt 1380

cggaggagag ggcccgagaa ggggccatac cagggcgcgg cgctgggttg ccacacttgg 1440

gaaagcagcc cggagctggg tgctggggaa ggcggggcgc gtagcctccc gccgccctgc 1500

ggttgggccg gtggaggccc aggcgttgct aggattgcat cagttttcct gtttgcacta 1560

tttctttttg taacattggc cctgtgtgaa gtatttcgaa tctcctcctt gctctgaaac 1620

ttcagcgatt ccattgtgat aagcgcacaa acagcactgt ctgtcggtaa tcggtactac 1680

tttattaatg attttctgtt acactgtata gtagtcctat ggcaccccca ccccatccct 1740

ttcgtgccac tcccgtcccc acccccaccc cagtgtgtat aagctggcat ttcgccagct 1800

tgtacgtagc ttgccactca gtgaaaataa taacattatt atgagaaagt ggacttaacc 1860

gaaatggaac caactgacat tctatcgtgt tgtacataga atgatgaagg gttccactgt 1920

tgttgtatgt cttaaattta tttaaaactt tttttaatcc agatgtagac tatattctaa 1980

aaaataaaaa agcaaatgtg tcaactaaat tggacaagcg tctggtcctc attaatctgc 2040

caatgaatgg tttcgtcatt aaataaaaat caatttaatt gatttactag ca 2092

<210> SEQ ID NO 2

<211> LENGTH: 183

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 5595953

<400> SEQUENCE: 2

Met Met Gln Ile Cys Asp Thr Tyr Asn Gln Lys His Ser Leu Phe

1 5 10 15

Asn Ala Met Asn Arg Phe Ile Gly Ala Val Asn Asn Met Asp Gln

20 25 30

Thr Val Met Val Pro Ser Leu Leu Arg Asp Val Pro Leu Ala Asp

35 40 45

Pro Gly Leu Asp Asn Asp Val Gly Val Glu Val Gly Gly Ser Gly

50 55 60

Gly Cys Leu Glu Glu Arg Thr Pro Pro Val Pro Asp Ser Gly Ser

65 70 75

Ala Asn Gly Ser Phe Phe Ala Pro Ser Arg Asp Met Tyr Ser His

80 85 90

Tyr Val Leu Leu Lys Ser Ile Arg Asn Asp Ile Glu Trp Gly Val

95 100 105

Leu His Gln Pro Pro Pro Pro Ala Gly Ser Glu Glu Gly Ser Ala

110 115 120

Trp Lys Ser Lys Asp Ile Leu Val Asp Leu Gly His Leu Glu Gly

125 130 135

Ala Asp Ala Gly Glu Glu Asp Leu Glu Gln Gln Phe His Tyr His

140 145 150

Leu Arg Gly Leu His Thr Val Leu Ser Lys Leu Thr Arg Lys Ala

155 160 165

Asn Ile Leu Thr Asn Arg Tyr Lys Gln Glu Ile Gly Phe Gly Asn

170 175 180

Trp Gly His

<210> SEQ ID NO 3

<211> LENGTH: 502

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 181, 317, 363, 393, 402, 421, 432, 458, 461, 463, 470,

478, 482, 494

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 1479946F6

<400> SEQUENCE: 3

gggcctttta tctcggtgct gccgggggag gcgggaggag gagacaccag gggtggccct 60

gagcgccggc gacacctttc ctggactata aattgagcac ctgggatggg tagggggcca 120

acgcatcacc gccgtccgca gtcacagtcc agccactgac cgcagcagcg cccttgcgta 180

nagccgcttg cagcgagaac actgaattgc caacgagcag gagagtctca aggcgcaaga 240

ggaggccagg ggctcgaccc acagagcacc ctcagccatc gcgagtttcc gggcgccaaa 300

gccaggagaa gccgccnatc ccgcaaggcc cggtctgcca gcgagacgag attggcgagg 360

gcngaagagt gccgggaatc ccgccacacc ggntatagca ancccccagc gcgggctttg 420

naaacgcctg angcgggcat cccttgaccg gcgacatncc ntnccctgcn tcctgggntc 480

ancttcgggc gcancatatt ac 502

<210> SEQ ID NO 4

<211> LENGTH: 529

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 48, 172, 214, 348, 364, 414, 417, 428, 430, 436, 471,

491, 495, 503, 511, 523

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 3241390F6

<400> SEQUENCE: 4

gcgagtttcc gggcgccaaa gccaggagaa gccgcccatc ccgcaggnca ggtctgccag 60

cgagacgaga gttggcgagg gcggaggagt gccgggaatc ccgccacacc ggctatagcc 120

aggcccccag cgcgggcctt ggagagcgcg tgaaggcggg catccccttg anccggccga 180

ccatccccgt gcccctgcgt ccctgcgctc caangtccgc gcggccacca tgatgcaaat 240

ctgcgacacc tacaaccaga agcactcgct ctttaacgcc atgaatcgct tcattggcgc 300

cgtgaacaac atggaccaga cggtgatggt gcccagcttg tgcgcgangt gcccctggct 360

gacnccgggt tagacaacga tgttggcgtg gaggtaagcg gcaatggcgg cttnctngag 420

gagcgcangn ccccanttcc cgactcggga agcgccaatg gagctttttt nggggcctct 480

tggggacaat nttanaagcc aantaagtgg nttctcaaag ttncatccg 529

<210> SEQ ID NO 5

<211> LENGTH: 562

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 288, 289, 291, 293, 313, 434, 514, 560

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 1432520R1

<400> SEQUENCE: 5

gacggtgatg gtgcccagct tgctgcgcga cgtgcccctg gctgaccccg ggttagacaa 60

cgatgttggc gtggaggtag gcggcagtgg cggctgcctg gaggagcgca cgcccccagt 120

ccccgactcg ggaagcgcca atggcagctt tttcgcgccc tctcgggaca tgtacagcca 180

ctacgtgctt ctcaagtcca tccgcaacga catcgagtgg ggggtcctgc accagccgcc 240

tccaccggct gggagcgagg agggcagtgc ctggaagtcc aaggacannc ngntggacct 300

gggccacttg ganggtgcgg acgccggcga agaagacctg gaacagcagt tccactacca 360

cctgcgcggg ctgcacactg tgtctcgaaa ctcacgcgca aagccaacat cctcactaac 420

agtacaagca ggantcggtt cggaattggg ggcactgagg cgtggcgccc gtggctgccc 480

agaacttttc gaccatctaa cctctctatt cctnaagctt tttttttttc cggctggggg 540

cggaaggcaa ctgcaaattn gg 562

<210> SEQ ID NO 6

<211> LENGTH: 254

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 80, 215, 238, 239, 242, 243, 244, 249

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 4534217H1

<400> SEQUENCE: 6

gggcagactg caaactgggg ggctgcgtac gtgcaggagg cgcggtgggg ctgcgtggag 60

gagggggcca cgtgtgagan agaagaaaat ggtggccgga gatgggaggg cccaaggaac 120

ctcctgggag ggggcctgca ttctatgttg gtgggaatgg gactgggctg acgccctgca 180

ttcagcctgt gcctttcctg gggtttcttt tctgntcttt tcggaggaga aggcccgnna 240

annngccana ccaa 254

<210> SEQ ID NO 7

<211> LENGTH: 238

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 19, 133

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 2191992H1

<400> SEQUENCE: 7

cgcggtgggg ctgcgtggng gagggggcca cgtgtgagag agaagaaaat ggtggccgga 60

gatgggaggg cccaaggaac ctcctgggag ggggcctgca ttctatgttg gtgggaatgg 120

gactgggctg acnccctgca ttcagcctgt gcctttcctg gggtttcttt tctgttcttt 180

tcggaggaga gggcccgaga aggggccata ccagggcgcg gcgctgggtt gccacact 238

<210> SEQ ID NO 8

<211> LENGTH: 661

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 2, 5, 65, 68, 70, 75, 96, 104, 107, 110, 116, 131, 194,

195, 198, 199, 200, 204, 225, 227, 235, 309, 469, 534, 536, 559,

563, 591, 603, 604, 612, 619, 632, 657

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 1320132T1

<400> SEQUENCE: 8

tnganttaaa aaaagtttta aataaattta agacatacca acaacagtgg aaaccttcat 60

cattntangn acaanacgat agaatgtcag ttgggnccat ttcngtnaan tccacnttct 120

cataataatg ntattatttt cactgagtgg caagctacgt acaagctggc gaaatgccag 180

cttatacaca ctgnngtnnn ggtngggacg ggagtggcac gaaangnatg gggtnggggt 240

gccataggac tactatacag tgtaacagaa aatcattaat aaagtagtac cgattaccga 300

cagacagtnc tgtttgtgcg cttatcacaa tggaatcgct gaagtttcag agcaaggagg 360

agattcgaaa tacttcacac agggccaatg ttacaaaaag aaatagtgca aacaggaaaa 420

ctgatgcaat cctagcaacg cctgggcctc caccggccca accgcaggnt gcgggaggct 480

acgcgccccg ccttccccag cacccagctc cgggctgctt tcccaagtgt tgcnanccaa 540

cgccgcgccc tggtattgnc ccntctcggg cctttcctcc gaaaagaacc ngaaagaacc 600

ccnngaaagg cncaggctna attcagggcg tnacccagtt ccattcccac caacttngat 660

t 661

<210> SEQ ID NO 9

<211> LENGTH: 624

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: (302)...(307), 330, 332, 334, 560, 572, 574, 577, 593,

609

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 1516707T1

<400> SEQUENCE: 9

atttttattt aatgacgaaa ccattcattg gcagattaat gaggaccaga cgcttttcca 60

atttagttga cacatttgct tttttatttt ttagaatata gtctacatct ggattaaaaa 120

aagttttaaa taaatttaag acatacaaca acagtggaac ccttcatcat tctatgtaca 180

acacgataga atgtcagttg gttccatttc ggttaagtcc actttctcat aataatgtta 240

ttattttcac tgagtggcaa gctacgtaca agctggcgaa atgccagctt atacacactg 300

gnnnnnnggt ggggacggga gtggcacgan angnatgggg tgggggtgcc ataggactac 360

tatacagtgt aacagaaaat cattaataaa gtagtaccga ttaccgacag acagtgctgt 420

ttgtgcgctt atcacaatgg aatcgctgaa gtttcagagc aaggaggaga ttcgaaatac 480

ttcacacagg gccaatgtta caaaaagaaa tagtgcaaac aggaaaactg atgcaatcct 540

agcaacgcct gggcctccan cggcccaacc gnangcngcg ggaggctacg cgncccgctt 600

ccccagcanc cagctccggg gtgt 624

<210> SEQ ID NO 10

<211> LENGTH: 252

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 186, 189, 195, 196, 200, 204, 208, 213, 222, 226, 228,

236, 244, 248, 251

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 5595953H1

<400> SEQUENCE: 10

atttcgaatc tcctccttgc tctgaaactt cagcgattcc attgtgataa gcgcacaaac 60

agcactgtct gtcggtaatc ggtactactt tattaatgat tttctgttac actgtatagt 120

agtcctatgg cacccccacc ccatcccttt cgtgccactc ccgtccccac ccccacccca 180

gggggntang cgggnntttn gccngctnga cgnagctggc cnctcngnga aaatantacc 240

tttnttgngg ng 252

<210> SEQ ID NO 11

<211> LENGTH: 302

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 89, 186, 251, 252, 254, 255, 258, 259, 260, 267, 268,

278, 281, 283, 286, 291, 292, 294, 297

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 1988906R6

<400> SEQUENCE: 11

ggacttaacc gaaatggaac caactgacat tctatcgtgt tgtacataga atgatgaagg 60

gttccactgt tgttgtatgt cttaaattna tttaaaactt tttttaatcc agatgtagac 120

tatattctaa aaaataaaaa agcaaatgtg tcaactaaat tggacaagcg tctggtcctc 180

attaanctgc caatgaatgg tttcgtcatt aaataaaaat caatttaatt gatttactag 240

caaaagtaga nnannaannn aaaaaannaa aaaaaaanac naangntaac nntnccnaaa 300

aa 302

<210> SEQ ID NO 12

<211> LENGTH: 144

<212> TYPE: DNA

<213> ORGANISM: Macaca fascicularis

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 25

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 700712962H1

<400> SEQUENCE: 12

ctcgatgctg ccgggggagg cgggnggagg agacaccagg ggtggccctg agcaccggcg 60

acacctttcc tggactataa attgagcacc tgggatgggt agggggtcaa cgcatcaccg 120

ccgcccgcag tcacagtccg gcca 144

<210> SEQ ID NO 13

<211> LENGTH: 274

<212> TYPE: DNA

<213> ORGANISM: Macaca fascicularis

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 19, 253

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 700715135H1

<400> SEQUENCE: 13

gacgccaaag ccaggagang ccgcccatcc cgcaggtccg gttctgccag cgagacgaga 60

gttggcgagg gcggaggagt gccgggaatc ccgccacacc ggctatagcc aggcccccag 120

cgcgggcctt ggagagagcg tgaaggcggg catccctttg acccggccga ccatccccgt 180

gtctctgcgt ccctgcgctc cagcgcccgc gcggccacca tgatgcaaat ctgcgacacc 240

tacaaccaga agnactcgct ctttaacggc atga 274

<210> SEQ ID NO 14

<211> LENGTH: 273

<212> TYPE: DNA

<213> ORGANISM: Mus musculus

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 245

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 701253541H1

<400> SEQUENCE: 14

cccgggtaca tgtacagcca ctacgtgctg ctcaagtcca tccgcaatga tatcgagtgg 60

ggagtcctgc accagccttc gtctccgccg gccgggagcg aggagagcac ctggaagccc 120

aaggacatcc tggtgggcct gagtcacttg gagagcgcgg atgcggcgag gaagatctgg 180

agcagcagtt ccactaccac ctgcgcgggc tgcacaccgt gctctccaaa ctcacccgaa 240

aagcnaacat cctcaccatt agatacaagc agg 273

<210> SEQ ID NO 15

<211> LENGTH: 250

<212> TYPE: DNA

<213> ORGANISM: Mus musculus

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 45

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 701252210H1

<400> SEQUENCE: 15

ctttttgtaa cagtgaccct gtcttaagtc tttcagatct ctttnctttg aaacttcgtc 60

gattccattg tgataagcgc acaaacagca ctgttggtaa ccggtactac tttattaatg 120

attttctgtt acactgtaca gtagtcctgt ggcaccctat ccctttcacg ccacccctcc 180

cccgcccgtg tgtgtaaact ggcgatgtgc cagctaggat gaagcttgcc actcggctag 240

cgaaaataat 250

<210> SEQ ID NO 16

<211> LENGTH: 272

<212> TYPE: DNA

<213> ORGANISM: Rattus norvegicus

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 56, 64, 67, 84, 209, 234, 249

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 700545683H1

<400> SEQUENCE: 16

gaccttttat ctgtgctgct ggaggaggta ggaggaggag acatcagggg tggtcntggg 60

gcgnctngga cacctatcct ggantataaa ttgagcacct gggatgcagc agggggccga 120

agcagccacc atcacccata ctcacagtcc gatcagtgac cgcagcagcg cccttgggca 180

gccaccgtgc cgcaactacg agcactgana accaggggat ttcgcagtgc aagngatcaa 240

ggctagacnc aaccacctac catcctcgtg ag 272

<210> SEQ ID NO 17

<211> LENGTH: 257

<212> TYPE: DNA

<213> ORGANISM: Rattus norvegicus

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 700145292H1

<400> SEQUENCE: 17

gcaactcgag cactgagaac caggggattt cgcagtgcaa gagatcaagg ctagacccaa 60

ccacctaaca tcctcgtgag ccaaagctta gagcagccgc gcatcaggaa gggctgaact 120

gagacagaag gaagagttag agagggcgga gaaggatctg ggaatccagt cacaccggct 180

tcaagcaggc tcccggcatt agcgtttgaa ggcgggcatc gccagaggtc tatctcggtg 240

taccagtgtc cctgtgt 257

<210> SEQ ID NO 18

<211> LENGTH: 239

<212> TYPE: DNA

<213> ORGANISM: Rattus norvegicus

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 16, 68

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 700861443H1

<400> SEQUENCE: 18

gacagaagga agagtnagag agggcggaga aggatctggg aatccagtca caccggcttc 60

aagcaggntt cccggcatta gcgtttgaag gcgggcatcg ccagaggtct atctcggtgt 120

accagtgtcc ctgtgtttcc gcgcccgctc ggccaccatg atgcaaatct gcgacacata 180

caaccagaag cactcgctct ttaacgccat gaatcgcttc attggcgcgg tgaacaaca 239

<210> SEQ ID NO 19

<211> LENGTH: 302

<212> TYPE: DNA

<213> ORGANISM: Rattus norvegicus

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 700225363H1

<400> SEQUENCE: 19

gtccctgtgt ttccgcgccc gctcggccac catgatgcaa atctgcgaca catacaacca 60

gaagcactcg ctctttaacg ccatgaatcg cttcattggc gcggtgaaca acatggacca 120

gacggtgatg gtgcccagtc tgctgcgcga tgtacccctg tccgagccgg atctagacaa 180

cgaggtcagc gtggaggtag gcggcagtgg cagctgcctg gaggagcgca cgaccccggc 240

cccaagcccg ggcagcgcca atggaagctt tttcgcgccc tcccgggaca tgtacagcca 300

ct 302

<210> SEQ ID NO 20

<211> LENGTH: 286

<212> TYPE: DNA

<213> ORGANISM: Rattus norvegicus

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 62, 283

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 700643425H1

<400> SEQUENCE: 20

ggaccagacg gtgatggtgc ccagtctgct gcgcgatgta cccctgtccg agccggatct 60

anacaacgag gtcagcgtgg aggtaggcgg cagtggcagc tgcctggagg agcgcacgac 120

cccggcccca agcccgggca gcgccaatgg aagctttttc gcgccctccc gggacatgta 180

cagccactac gtgctgctca agtccatccg caacgatatt gagtggggag tcctgcacca 240

gccttcgtcc ccgccggctg ggagtgagga gggcacctgg aanccc 286

<210> SEQ ID NO 21

<211> LENGTH: 285

<212> TYPE: DNA

<213> ORGANISM: Rattus norvegicus

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 72, 103, 154, 172, 224, 263, 264, 283

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 700525920H1

<400> SEQUENCE: 21

gtgctgctca agtccatccg caacgatatt gagtggggag tcctgcacca gccttgcgtc 60

cccgccggct gngagtgagg agtggcacct ggaagcccaa ggncatcctg gtgggcctga 120

gccacttgga gagcacggat gcgggcgagg aagntctgga gcagcagttc cnctaccacc 180

tgcgcgggct gcacaccgtg ctctccaaac tcacccgcaa agcnaacatc cttaacaaca 240

gatacaagca ggagatcggc ttnntaatgg gggccattga ggngg 285

<210> SEQ ID NO 22

<211> LENGTH: 270

<212> TYPE: DNA

<213> ORGANISM: Rattus norvegicus

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 87

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 700773927H1

<400> SEQUENCE: 22

ggccaacatc cttaccaaca gatacaagca ggagatcggc ttcagtaatt ggggccactg 60

aggcggggtt gtccccgctg cccagcnccc tctcgggtcg gctctaccac ccccctctct 120

ttcctccaaa ctattttctt cctggttgtg gggcgcgaag ggcacgctgt aaagttgggc 180

tgtgtacttg gtggggtttg tgtggagaaa acagagcaga gagcagagga aatatcgcca 240

gagagggggg ttcaaagacc cccggagggc 270

<210> SEQ ID NO 23

<211> LENGTH: 283

<212> TYPE: DNA

<213> ORGANISM: Rattus norvegicus

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 700513679H1

<400> SEQUENCE: 23

ggaactgggc cgatgtcctt cattcagcct gtgcctttct tggggtttct tttctctttt 60

tctttccgga agagaagggc ctgagaaagg gccatgccag ggcacagcgc tgggttgcca 120

cacttgggag ggcagcttct agctgggtgc tcgggggagg cggggcacag cctcctgccc 180

gccctgcttt gagctgcaag aggaggcctt ggcgttgcta ggattgcgtc agttttcctg 240

tttgcactat ttctttttgt aacagtgacc ctgtcttaag tat 283

<210> SEQ ID NO 24

<211> LENGTH: 266

<212> TYPE: DNA

<213> ORGANISM: Rattus norvegicus

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 700767486H1

<400> SEQUENCE: 24

tttcagatct ttttgctttg aaacttcgtc gattccattg tgataagcgc acaagcagca 60

ctgttggtaa ccggtactac tttattaatg attttctgtt acactgtaca gtagtcctat 120

ggcaccccat ccctttcacg ccacccctcc cccaccccgt gtgtgtaaac tggtgacgtg 180

ccagctagga tgaagcttgc cactcggcca gcgaaaataa taacattatt gtgagaaagt 240

ggatttatct aaagtggaac caactg 266

<210> SEQ ID NO 25

<211> LENGTH: 199

<212> TYPE: DNA

<213> ORGANISM: Rattus norvegicus

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 8, 36, 40

<223> OTHER INFORMATION: a or g or c or t, unknown, or other

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: Incyte ID No.: 700327166H1

<400> SEQUENCE: 25

agccactngg ccagcgaaaa taataacatt attgtnagan agtggattta tctaatggaa 60

ccaactgaca ttctatctgt gttgtacgta gaatgatgaa gggctccact gttgttatat 120

gtcttgttta tttaaaactt ttttttaatc cagatgtaga ctatattcta aaaaataaaa 180

gctcagatgt gttaaccac 199

<210> SEQ ID NO 26

<211> LENGTH: 152

<212> TYPE: PRT

<213> ORGANISM: Danio rerio

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: GenBank ID No: g861207

<400> SEQUENCE: 26

Met Gln Met Ser Glu Pro Leu Ser Gln Lys Asn Ala Leu Tyr Thr

1 5 10 15

Ala Met Asn Arg Phe Leu Gly Ala Val Asn Asn Met Asp Gln Thr

20 25 30

Val Met Val Pro Ser Leu Leu Arg Asp Val Pro Leu Asp Gln Glu

35 40 45

Lys Glu Gln Gln Lys Leu Thr Asn Asp Pro Gly Ser Tyr Leu Arg

50 55 60

Glu Ala Glu Ala Asp Met Tyr Ser Tyr Tyr Ser Gln Leu Lys Ser

65 70 75

Ile Arg Asn Asn Ile Glu Trp Gly Val Ile Arg Ser Glu Asp Gln

80 85 90

Arg Arg Lys Lys Asp Thr Ser Ala Ser Glu Pro Val Arg Thr Glu

95 100 105

Glu Glu Ser Asp Met Asp Leu Glu Gln Leu Leu Gln Phe His Leu

110 115 120

Lys Gly Leu His Gly Val Leu Ser Gln Leu Thr Ser Gln Ala Asn

125 130 135

Asn Leu Thr Asn Arg Tyr Lys Gln Glu Ile Gly Ile Ser Gly Trp

140 145 150

Gly Gln

<210> SEQ ID NO 27

<211> LENGTH: 150

<212> TYPE: PRT

<213> ORGANISM: Mus musculus

<220> FEATURE:

<221> NAME/KEY: misc_feature

<223> OTHER INFORMATION: GenBank ID No: g1171574

<400> SEQUENCE: 27

Met Gln Val Leu Thr Lys Arg Tyr Pro Lys Asn Cys Leu Leu Thr

1 5 10 15

Val Met Asp Arg Tyr Ser Ala Val Val Arg Asn Met Glu Gln Val

20 25 30

Val Met Ile Pro Ser Leu Leu Arg Asp Val Gln Leu Ser Gly Pro

35 40 45

Gly Gly Ser Val Gln Asp Gly Ala Pro Asp Leu Tyr Thr Tyr Phe

50 55 60

Thr Met Leu Lys Ser Ile Cys Val Glu Val Asp His Gly Leu Leu

65 70 75

Pro Arg Glu Glu Trp Gln Ala Lys Val Ala Gly Asn Glu Thr Ser

80 85 90

Glu Ala Glu Asn Asp Ala Ala Glu Thr Glu Glu Ala Glu Glu Asp

95 100 105

Arg Ile Ser Glu Glu Leu Asp Leu Glu Ala Gln Phe His Leu His

110 115 120

Phe Cys Ser Leu His His Ile Leu Thr His Leu Thr Arg Lys Ala

125 130 135

Gln Glu Val Thr Arg Lys Tyr Gln Glu Met Thr Gly Gln Val Leu

140 145 150

Claims

What is claimed is:

1. An isolated polypeptide selected from the group consisting of:

a) a polypeptide comprising an amino acid sequence of SEQ ID NO:2,

b) a polypeptide comprising a naturally occurring an amino acid sequence at least 90% identical to an amino acid sequence of SEQ ID NO:2,

c) a biologically active fragment of a polypeptide having an amino acid sequence of SEQ ID NO:2, and

d) an immunogenic fragment of a polypeptide having an amino acid sequence of SEQ ID NO:2.

2. An isolated polypeptide of claim 1, comprising an amino acid sequence of SEQ ID NO:2.

3. An isolated polynucleotide encoding a polypeptide of claim 1.

4. An isolated polynucleotide encoding a polypeptide of claim 2.

5. An isolated polynucleotide of claim 4, having a sequence of SEQ ID NO:1.

6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3.

7. A cell transformed with a recombinant polynucleotide of claim 6.

8. A transgenic organism comprising a recombinant polynucleotide of claim 6.

9. A method of producing a polypeptide of claim 1, the method comprising:

a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and

b) recovering the polypeptide so expressed.

10. A method of claim 9, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:2.

11. An isolated antibody which specifically binds to a polypeptide of claim 1.

12. An isolated polynucleotide selected from the group consisting of:

a) a polynucleotide comprising a polynucleotide sequence of SEQ ID NO:1,

b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence of SEQ ID NO:1,

c) a polynucleotide complementary to a polynucleotide of a),

d) a polynucleotide complementary to a polynucleotide of b) and

e) an RNA equivalent of a)-d).

13. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim 12.

14. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising:

a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and

b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof.

15. A method of claim 14, wherein the probe comprises at least 60 contiguous nucleotides.

16. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising:

a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and

b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.

17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient.

18. A composition of claim 17, wherein the polypeptide comprises an amino acid sequence of SEQ ID NO:2.

19. A method for treating a disease or condition associated with decreased expression of functional LMTF, comprising administering to a patient in need of such treatment the composition of claim 17.

20. A method of screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising:

a) exposing a sample comprising a polypeptide of claim 1 to a compound, and

b) detecting agonist activity in the sample.

21. A composition comprising an agonist compound identified by a method of claim 20 and a pharmaceutically acceptable excipient.

22. A method for treating a disease or condition associated with decreased expression of functional LMTF, comprising administering to a patient in need of such treatment a composition of claim 21.

23. A method of screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising:

a) exposing a sample comprising a polypeptide of claim 1 to a compound, and

b) detecting antagonist activity in the sample.

24. A composition comprising an antagonist compound identified by a method of claim 23 and a pharmaceutically acceptable excipient.

25. A method for treating a disease or condition associated with overexpression of functional LMTF, comprising administering to a patient in need of such treatment a composition of claim 24.

26. A method of screening for a compound that specifically binds to the polypeptide of claim 1, the method comprising:

a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and

b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1.

27. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, said method comprising:

a) combining the polypeptide of claim 1 with at least one test compound under conditions permissive for the activity of the polypeptide of claim 1,

b) assessing the activity of the polypeptide of claim 1 in the presence of the test compound, and

c) comparing the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, wherein a change in the activity of the polypeptide of claim 1 in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide of claim 1.

28. A method of screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence of claim 5, the method comprising:

a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide,

b) detecting altered expression of the target polynucleotide, and

c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.

29. A method of assessing toxicity of a test compound, the method comprising:

a) treating a biological sample containing nucleic acids with the test compound,

b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof,

c) quantifying the amount of hybridization complex, and

d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.

30. A diagnostic test for a condition or disease associated with the expression of LMTF in a biological sample, the method comprising:

a) combining the biological sample with an antibody of claim 11, under conditions suitable for the antibody to bind the polypeptide and form an antibody:polypeptide complex, and

b) detecting the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample.

31. The antibody of claim 11, wherein the antibody is:

a) a chimeric antibody,

b) a single chain antibody,

c) a Fab fragment,

d) a F(ab′)₂fragment, or

e) a humanized antibody.

32. A composition comprising an antibody of claim 11 and an acceptable excipient.

33. A method of diagnosing a condition or disease associated with the expression of LMTF in a subject, comprising administering to said subject an effective amount of the composition of claim 32.

34. A composition of claim 32, wherein the antibody is labeled.

35. A method of diagnosing a condition or disease associated with the expression of LMTF in a subject, comprising administering to said subject all effective amount of the composition of claim 34.

36. A method of preparing a polyclonal antibody with the specificity of the antibody of claim 11, the method comprising:

a) immunizing an animal with a polypeptide consisting of an amino acid sequence of SEQ ID NO:2, or an immunogenic fragment thereof, under conditions to elicit an antibody response,

b) isolating antibodies from said animal, and

c) screening the isolated antibodies with the polypeptide, thereby identifying a polyclonal antibody which binds specifically to a polypeptide comprising an amino acid sequence of SEQ ID NO:2.

37. A polyclonal antibody produced by a method of claim 36.

38. A composition comprising the polyclonal antibody of claim 37 and a suitable carrier.

39. A method of making a monoclonal antibody with the specificity of the antibody of claim 11, the method comprising:

b) isolating antibody producing cells from the animal,

c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells,

d) culturing the hybridoma cells, and

e) isolating from the culture monoclonal antibody which binds specifically to a polypeptide comprising an amino acid sequence of SEQ ID NO:2.

40. A monoclonal antibody produced by a method of claim 39.

41. A composition comprising the monoclonal antibody of claim 40 and a suitable carrier.

42. The antibody of claim 11, wherein the monoclonal antibody is produced by screening a Fab expression library.

43. The antibody of claim 11, wherein the antibody is produced by screening a recombinant immunoglobulin library.

44. A method of detecting a polypeptide comprising an amino acid sequence of SEQ ID NO:2 in a sample, the method comprising:

a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and

b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide comprising an amino acid sequence of SEQ ID NO:2 in the sample.

45. A method of purifying a polypeptide comprising an amino acid sequence of SEQ ID NO:2 from a sample, the method comprising:

b) separating the antibody from the sample and obtaining the purified polypeptide comprising an amino acid sequence of SEQ ID NO:2.

46. A microarray wherein at least one element of the microarray is a polynucleotide of claim 12.

47. A method of generating an expression profile of a sample which contains polynucleotides, the method comprising:

a) labeling the polynucleotides of the sample,

b) contacting the elements of the microarray of claim 46 with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybridization complex, and

c) quantifying the expression of the polynucleotides in the sample.

48. An array comprising different nucleotide molecules affixed in distinct physical locations on a solid substrate, wherein at least one of said nucleotide molecules comprises a first oligonucleotide or polynucleotide sequence specifically hybridizable with at least 30 contiguous nucleotides of a target polynucleotide, and wherein said target polynucleotide is a polynucleotide of claim 12.

49. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 30 contiguous nucleotides of said target polynucleotide.

50. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 60 contiguous nucleotides of said target polynucleotide.

51. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to said target polynucleotide.

52. An array of claim 48, which is a microarray.

53. An array of claim 48, further comprising said target polynucleotide hybridized to a nucleotide molecule comprising said first oligonucleotide or polynucleotide sequence.

54. An array of claim 48, wherein a linker joins at least one of said nucleotide molecules to said solid substrate.

55. An array of claim 48, wherein each distinct physical location on the substrate contains multiple nucleotide molecules, and the multiple nucleotide molecules at any single distinct physical location have the same sequence, and each distinct physical location on the substrate contains nucleotide molecules having a sequence which differs from the sequence of nucleotide molecules at another distinct physical location on the substrate.