CA2437960A1 - Als2 gene and amyotrophic lateral sclerosis type 2 - Google Patents

Abstract

Candidate gene causing Amyotrophic Lateral Sclerosis type 2 (ALS2), encoding a putative GTP exchange factor (GEF) or GTPase regulatory factor.

Description

Related Applications This application claims priority from United States application no. 60/267,723 filed S February 12, 2001; Japanese application no. 2001-116973 filed April 16, 2001; and, United States application no. 60/318,352 filed September 12, 2001, which applications are hereby incorporated by reference.
Field of the Invention This invention relates to genetic causes of amyotrophic lateral sclerosis of type 2 ("ALS2").
Backeround of the Invention Amyotrophic lateral sclerosis ("ALS") is a progressive neurodegenerative disease in which distal and proximal motor neurons are selectively degenerated'. Its cause is ambiguous and its onset is mostly at middle age and thereafter. Its rate of onset is about 2-6 per 100,000 persons and begins with lowering of muscular strength and myoatrophy of wrist muscle as a secondary neuron hindrance resulting in bulbar paralysis symptoms such as atrophy of muscle of limbs, atrophy of tongue, alalia, dysphagia and dyspnea.
No therapeutic method has been established yet and most of the afflicted die within five years from onset.
Juvenile amyotrophic lateral sclerosis of type 2 ("ALS2"; OMIM2151002) is a somatically recessive type hereditary disease. Although the frequency of its onset is rare, muscular convulsion of limbs, face and throat is gradually expressed in persons of teens or twenties and becomes chronic by bulbar paralysis as described above.
Amyotrophic lateral sclerosis of type 2 has been mapped to the 1.7 cM interval flanked by D2SI16 and D2S2237 on human chromosome 2q333'4. Alterations in 391 exons and their flanking regions derived from 43 non-overlapping transcripts have been noted within this intervals'6.
ALS is a very severe disease and there is a need for development of means for its early detection or diagnosis and for treatment.
SUBSTITUTE SHEET (RULE 26) Summary of the Invention We have now identified a gene associated with amyotrophic lateral sclerosis type 2, termed the ALS2 or ALS2CR6 gene. This gene is expressed in various human tissues including neurons in the brain and spinal cord, and encodes a protein with homology to RanGED and RhoGEF.
This invention now provides mammalian ALS2 genes and mutant versions thereof as well as peptides (including proteins) encoded by such genes. Also included are fragments and nucleic acids derived from these genes, corresponding peptides, and oligonucleotides suitable for use as amplification primers and/or probes. Antibodies to the peptides of this invention are also provided.
This invention also provides methods of diagnosis of ALS2 which may include identifying in a patient at risk, an altered ALS2 gene or protein. The patient may be tested to characterize one or more mutations in the gene or protein produced. Such a mutation may comprise the A261de1 mutation or the AGI548de1 mutations described herein.
This invention also provides nucleic acids which correspond to a region of the gene, which nucleic acids typically hybridize to at least about 6, at least about 10, at least about 15, at least about 20, or at least about 25 consecutive nucleotides of an ALS2 sequence as described herein, or to complements of such sequences, or to naturally occurring mutants or allelic variants thereof. The probes or primers may be chosen to be capable of distinguishing (such as by amplification or hybridization) allelic variants, including the A261 del and AGI548de1 mutations described herein. Such probes or primers may fiuther include a label which is capable of being detected. This invention also provides kits for identifying ALS2 genes, including those comprising alleles associated with an ALS2 disease state, wherein the kits may comprise a probe or primer as described herein.
The kit may further comprise instructions for using the probes or primers to distinguish alleles as described herein.
This invention also provides vectors containing nucleic acids of this invention, including vectors adapted for expression of such nucleic acids in a target cell or organism.
Such vectors may comprise appropriate transcription regulatory elements for directing transcription of the nucleic acids in a target cell or organism. Nucleic acids and peptides of this invention may be expressed in bacterial as well as eukaryotic cells, including SUBSTITUTE SHEET (RULE 26) mammalian cells. Such vectors may be adapted to express nucleic acids of this invention in a reverse direction so as to generate anti-sense transcription products.
This invention also provides non-human mammals comprising a genome in which an ALS2 gene has been mutated, including by deletion. Such a mammal may be a mouse and S methods for altering the murine genome such as to produce an ALS2 "knock-out" mouse, are described herein and are known in the art.
This invention also provides the use of nucleic acids and peptides as disclosed herein for the preparation of medicaments for treatment of ALS2 or in the treatment of ALS2.
This invention also provides methods of treating patients for ALS2, which methods may comprise testing the patient to diagnose or characterize an ALS2 disease state. A
patient may be treated for ALS2, for example by administering to the patient or by otherwise providing a native form or functional fragment or derivative of the ALS2 peptide described herein or such other therapeutic agent as which will restore function of the protein in a patient. Also included in this invention are vectors suitable for use in gene therapy and gene therapy methodologies whereby a patient is treated to restore the function of ALS2 by delivering or producing a functional gene for expression in the patient. Gene therapy vectors may, for example, be adeno-associated vector, such as those known in the art.
General methods for gene therapy are also known in the art.
This invention includes a human ALS2 gene which is present in human second chromosome q33 region and may code for a GTPase regulatory factor. The gene may encode an amino acid sequence of SEQ ID N0:2. cDNA synthesized from mRNA that may be transcribed by this gene has a base sequence of SEQ ID NO:1.
This invention includes a human ALS2 mutated gene which is related to amyotrophic lateral sclerosis of type 2 and codes for a modified protein having an amino acid sequence of SEQ ID NO: 3 or SEQ ID N0:84, by a deficiency of one or two bases of the above human ALS2 gene.
This invention includes nucleic acids purified from genomic DNA, mRNA or cDNA
as well as synthesized nucleic acids.
This invention includes oligonucleotides which hybridize to ALS2 genes and variants thereof, preferably under.stringent conditions.
SUBSTITUTE SHEET (RULE 26) This invention includes kits comprising oligonucleotides or oligonucleotide primer sets which may be used to carry out amplification of ALS2 encoded nucleic acids, for example by the polymerase chain reaction (PCR).
This invention includes oligonucleotide probes which hybridize to the regions containing base deficient sites (A261de1 and AGI548de1J in ALS2 under stringent conditions.
This invention includes oligonucleotide primer sets which carry out a PCR
amplification of the region containing a base deficient site in ALS2 as described herein. A
specific example of this primer set is a pair of synthetic oligonucleotides comprising the base sequences of SEQ ID NO: 6 and NO: 7 or a pair of synthetic oligonucleotides comprising the base sequences of SEQ ID NO: 8 and NO: 9.
This invention includes recombinant vectors comprising the above nucleic acids and cells transcribed by said recombinant vectors.
This invention includes a GTPase regulatory factor or a GEF which is characterized in being an expression product of an ALS2 gene as described herein.
Embodiments of such GTPase regulatory factors of GEF's are recombinant proteins produced by the transformed cells transformed according to this invention..
This invention includes a peptide comprising an amino acid sequence having continuous 5 or more acid amino residues in the first to the 46th amino acid sequence in SEQ ID NO: 2 and also a peptide comprising an amino acid sequence having continuous 5 or more acid amino residues in the 47th to the 1657th amino acid sequence in SEQ ID NO:
2. These peptides may be used for production of antibodies.
This invention also provides a modified protein which may be an expression product of a mutant human ALS2 gene and which comprises the amino acid sequence of SEQ
ID
NO: 3. An embodiment of this modified protein is a recombinant protein produced by a transformed cell.
This invention includes an antibody which recognizes peptides (including proteins) as disclosed herein. Embodiments of this antibody are an antibody which is prepared using a peptide according to this invention as an antigen, including a peptide comprising an amino acid sequence having continuous 5 or more acid amino residues in the first to the 46th amino acid sequence in SEQ ID NO: 2 and also an antibody which is prepared using a peptide SUBSTITUTE SHEET (RULE 26) comprising an amino acid sequence having continuous S or more acid amino residues in the 47th to the 1657th amino acid sequence in SEQ ID NO: 2 as an antigen.
This invention furthermore provides methods for the diagnosis of amyotrophic lateral sclerosis of type 2 which is characterized in detecting ALS2 mutated genes. An 5 embodiment of this method for the diagnosis it that genomic DNA of the cells of a person to be diagnosed is subjected to a PCR amplification using a primer set comprising a pair of synthetic oligonucleotides comprising the base sequences of SEQ ID NO: 6 and NO: 7 or a pair of synthetic oligonucleotides comprising the base sequences of SEQ ID NO:
8 and NO:
9, the resulting DNA fragments are treated with a restriction enzyme NarI and the said person where each of the DNA fragments is divided into two fragments is judged to be suffering from amyotrophic lateral sclerosis of type 2.
This invention also provides a method for the diagnosis of amyotrophic lateral sclerosis of type 2 which is characterized in that the transcribed product of an ALS2 gene or mutated gene is detected. In an embodiment of this diagnostic method, the transcribed product is cDNA or mRNA of the gene of an ALS2 mutated gene or the modified protein expressed by the said mutated gene. An embodiment of the case of detection of the modified protein is a method for the detection of the protein where the antibody recognizing the first to the 46th amino acid sequences in SEQ ID NO: 2 reacts but the antibody recognizing the 47th to the 1657th amino acid sequence region in SEQ ID NO: 2 does not react.
Further, this invention provides a mouse ALS2 gene which may have an amino acid sequence of SEQ ID NO:S as well as nucleic acids derived therefrom including nucleic acids synthesized or purified from genomic DNA, mRNA or cDNA of the mouse gene or a complementary sequence thereof.
This invention also provides a gene-defective non-human mammal such as a rodent, preferably a mouse, where function of an ALS2 gene is substantially deficient.
Also provided are tissues of such a mouse.
The human ALS2 gene according to this invention is a genomic gene which has 33 introns and 34 exons, exists in a genomic DNA of 80.3 kb adjacent to a polymorphic DNA
marker D2S2309 in human second chromosome q 33 region (refer to Fig. 1) and codes for a SUBSTITUTE SHEET (RULE 26) human GTPase regulatory factor having an amino acid sequence of SEQ ID N0:2.
In this ALS2 gene, its cDNA has a base sequence of SEQ ID NO: 1.
This invention provides an isolated nucleic acid that codes for a peptide having at least about 75, 80, 85, 90, 95, 97 or 100% identity to all of an amino acid sequence selected from the group consisting of SEQ ID N0:2; SEQ ID N0:3; SEQ ID N0:5; SEQ ID
N0:84; and, amino acids 372-1657 of SEQ ID N0:2. Also provided are the peptides encoded by these nucleic acids.
This invention also provides an isolated nucleic acid consisting essentially of a nucleotide sequence having at least about 75, 80, 85, 90, 95, 97 or 100%
identity to all of a nucleotide sequence or a complementary sequence thereof, selected from the group consisting of SEQ ID NO:1; SEQ ID N0:4; nucleotides 124-5094 of SEQ ID NO:1;
nucleotides 1225-5094 of SEQ ID NO:1; and, nucleotides 124-5076 of SEQ ID
N0:4. Also provided are the peptides encoded by these nucleic acids.
The nucleic acids of this invention may be joined to a second nucleic acid not naturally associated with the nucleic acid of this invention. By not naturally associated, it is meant that the second nucleic acid is not part of an ALS2 gene and is not directly joined to an ALS2 gene in the genome of a mammal.
This invention also provides an oligonucleotide of 6 to 75 nucleotides, wherein the oligonucleotide hybridizes to a nucleic acid of this invention or a complementary sequence thereof, under stringent conditions. An oligonucleotide of this invention may be joined to a label, which is any moiety suitable for detectable labelling of the nucleic acid or for binding of the nucleic acid to a non-nucleic acid moiety.
This invention also provides a peptide consisting essentially of a sequence of at least 5 contiguous amino acids from a sequence selected from the group consisting of amino acids 1-46 of SEQ ID N0:2; amino acids 47-1657 of SEQ ID N0:2; SEQ ID N0:3;
amino acids 43-49 of SEQ ID N0:3; SEQ ID N0:84; and amino acids 476 to 545 of SEQ ID
N0:84. These peptides are useful, for example in raising antibodies of this invention and for investigating the function of the ALS2 protein.
This invention also provides a non-human mammal comprising a mutated gene, wherein the gene but for the mutation would encode a protein having at least about 75, 80, 85, 90, 95, 97 or 100% sequence identity to all of SEQ ID N0:2 or SEQ ID N0:5.
SUBSTITUTE SHEET (RULE 26) This invention also provides a method for the diagnosis of amyotrophic lateral sclerosis type 2 in a patient, comprising detecting the presence of a mutation in a gene that encodes a protein having at least about 75, 80, 85, 90, 95, 97 or 100%
sequence identity to SEQ ID N0:2 in a patient or a biological sample from a patient.
This invention also provides a method for the diagnosis of amyotrophic lateral sclerosis type 2, comprising detecting the presence or absence of a protein having at least about 75, 80, 85, 90, 95, 97 or 100% sequence identity to all of SEQ ID N0:2 in a patient or a biological sample from a patient.
This invention also provides a method for the diagnosis of amyotrophic lateral sclerosis type 2, comprising detecting the presence or absence of a protein having at least about 75, 80, 85, 90, 95, 97 or 100% sequence identity to all of SEQ ID N0:3 or SEQ ID
N0:84 in a patient or a biological sample from a patient.
In the diagnostic methods of this invention, sequences may be compared to determine the presence of mutations; oligonucleotides may be used to detect hybridization to nucleic acids of the patient; amplification of nucleic acids of the patient may be performed;
proteins of the patient may be contacted with antibodies of this invention; or proteins produced in the patient may be evaluated for the function of ALS2 protein.
This invention also provides a method of treatment of amyotrophic lateral sclerosis type 2, comprising administering a peptide, a nucleic acid, or a pharmaceutical composition comprising the peptide or nucleic acid to a patient in need thereof, wherein the peptide comprises an amino acid sequence having at least about 75, 80, 85, 90, 95, 97 or 100%
identity to SEQ ID N0:2 or a fragment thereof, and wherein the nucleic acid codes for said peptide.
This invention also provides a method of treatment of amyotrophic lateral sclerosis type 2, comprising administering a composition to a patient in need thereof, wherein the composition mimics the biological activity of the peptide of SEQ ID NO. 2 or a fragment thereof.
This invention also provides the use of a peptide or a nucleic acid for preparation of a medicament for treatment of amyotrophic lateral sclerosis type 2, wherein the peptide comprises an amino acid sequence having at least about 75, 80, 85, 90, 95, 97 or 100%
identity to SEQ ID N0:2 or a fragment thereof, and the nucleic acid codes for said peptide.
SUBSTITUTE SHEET (RULE 26) In this specification the term "isolated" with reference to a nucleic acid or peptide means that a nucleic acid is separate from the genome of a cell, a peptide is separate from a cell but does not mean that the subject matter has been obtained from a genome or a cell. In some instances, nucleic acids and peptides of this invention may be synthesized using conventional techniques.
Two nucleic acid or protein sequences are considered substantially identical if, when optimally aligned, they share at least about 70% sequence identity. In alternative embodiments, sequence identity may for example be at least 75%, at least 90%
or at least 95%. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman,1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, U.S.A.). Sequence alignment may also be carned out using the BLAST algorithm, described in Altschul et al., 1990, J.
Mol. Biol. 215:403-10 (using the published default settings).
Nucleic acid sequences of the invention may in some embodiments be substantially identical, such as substantially identical gene targeting substrates and target sequences. The substantial identity of such sequences may be reflected in percentage of identity when optimally aligned that may for example be greater than 50%, 80% to 100%, at least 80%, at least 90% or at least 95%, which in the case of gene targeting substrates may refer to the identity of a portion of the gene targeting substrate with a portion of the target sequence, wherein the degree of identity may facilitate homologous pairing and recombination and/or repair. An alternative indication that two nucleic acid sequences are substantially identical is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, conditions. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHP04, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.2 x SSC/0.1% SDS at 42°C (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3).
Alternatively, SUBSTITUTE SHEET (RULE 26) hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65°C, and washing in 0.1 x SSC/0.1% SDS at 68°C (see Ausubel, et al. (eds), 1989, supra).
Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Technigues in Biochemistry and Molecular Biology --Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York).
Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
It is well known in the art that some modifications and changes can be made in the structure of a polypeptide without substantially altering the biological function of that peptide, to obtain a biologically equivalent polypeptide. In one aspect of the invention, LPL
S447X therapeutics may include peptides that differ from a portion of the wild-type LPL
sequence by conservative amino acid substitutions. As used herein, the term "conserved amino acid substitutions" refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution can be made without loss of function. In making such changes, substitutions of like amino acid residues can be made, for example, on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing.
In some embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydrophilicity value (e.g., within a value of plus or minus 2.0), where the following hydrophilicity values are assigned to amino acid residues (as detailed in United States Patent No. 4,554,101, incorporated herein by reference): Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2);
Gln (+p.2); Gly (0); Pro (-0.5); Thr (-0.4); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3); Phe (-2.5); and Trp (-3.4).
In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydropathic index (e.g., within a value of plus or minus 2.0). In such embodiments, each amino acid residue may be SUBSTITUTE SHEET (RULE 26) assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8);
Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gln (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, Val, Leu, Ile, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral:
Gly, Ser, Thr, 10 Cys, Asn, Gln, Tyr.
Brief DescriRtion of the Drawings Figure 1 is a transcription map of 3Mb region of human chromosome 2q33 including an ALS2 candidate region. The white open rectangle is between D2S116 to D2S2237z'3.
Positions of 7 STS markers, 12 polymorphic DNA markers and 42 independent transcription units are shown. Polarity of 38 transcription units are shown by arrows. The location of the ALS2 gene is designated "ALS2CR6" which term may be used interchangeably for below.
Figure 2 shows a process for the detection of ALS2 associated mutations. "a"
is an example of the Tunisian and Kuwaiti ALS2 families. Genotypes of the members constituting a family is shown based on previously reported results3'4. "b"
shows the result of sequence determination of mutation (A261 del) in genomic DNA of the Tunisian ALS2 family. Patient 10797 is A261 del of a homozygotic type and the carrier 10784 is a heterozygotic type. The sequence determination was carried out for PCR
products. "c"
shows the results of determination of mutation (AGI548de1) in the genomic DNA
in the Kuwaiti ALS2 family. Sequence of the reverse strand of exon 5 in the region of interest are shown. Individual 18279 is a normal sibling, who is unaffected by ALS2 and carries two normal haplotypes. The box in this sequence indicates the position of the bases deleted in affected members. Individual 18281 is an unaffected parent .who carnes one disease haplotype. The overlapping normal and mutated sequences are shown. Individual 18275 is SUBSTITUTE SHEET (RULE 26) affected and the figure shows a homozygous CT deletion in the reverse strand of exon 5:
The position of the deleted bases is indicated by the arrow. The corresponding forward sequence and coded normal amino acids and novel amino acids produced by frameshifting are indicated. "d" shows segregation of the A261de1 mutation in the Tunisian ALS2 family.
The presence of the deletion was assayed by the digestion with NarI, which only cuts mutated gene product. For exon-PCR products, the 339 by fragment representing the normal allele was cleaved into two fragments (225 by and 113 bp) in the mutant allele. For RT-PCR product, the 302 by product which represents the normal allele was cleaved into two fragments (195 by and 106 bp) in the mutant allele.
Figure 3 shows northern blot analysis of the ALS2 (ALS2CR6) mRNA. In "a", a northern blot containing 2 ~g of poly A+ mRNA of many adult human tissues is hybridized with exon 4 of ALS2 cDNA. In the lower drawings, the same blot is hybridized with human ~3-actin cDNA for confirmation of the property and the comparative load of RNA. In the left, size of the ALS2 transcript is shown. In "b", northern blot containing 10 pg of total RNA obtained normal whole brain and 20 ~g of total RNA obtained from lymphocytes of patients and healthy persons (10788 persons) was hybridized to exon 4 of the ALS2 cDNA.
The right panel shows an agarose gel electrophoresis of an RNA sample.
Figure 4 is a comparison of amino acid sequences in human ALS2CR6 and mouse homolog mALS2CR6. The same residues are shown by frames. There are shown the position of the additional three amino acid residues of the Tunisian mutant protein (starting from the 47th amino acid residue), the position of the 25 amino acid residues (starting from the 372nd residue) of a short variant part of the ALS2 gene and the position of the additional 70 amino acid residues of the Kuwaiti mutant protein (starting from the 476th residue).
Figure S shows an expression of ALS2 mRNA in brain and spinal cord of adult mouse. "a" is an arrow-like whole image of an RNA/RNA in situ hybridization using an antisense ALS2 riboprobe while "b" is a control image using a sense strand probe.
Significant expression was noted in neurons of hippocampus and dentate gyrus (c and g), Purkinje cells of cerebellum (d and h), neurons of cerebral cortex (e and i) and cinerea of spinal cord including anterior horn cells (f and j). A scale bar shows a length of 10 pm.
Figure 6 is a result of an amino acid sequence analysis. "a" is a schematic chart of domains and motifs in normal and mutated ALS2 protein. RCCl is a regulatory factor for SUBSTITUTE SHEET (RULE 26) chromosome condensation, DH is a homologous domain to Dbl, PH is a pleckstrin-homologous domain, MORN is membrane structure and recognition nexus and VPS9 is a vacuole protein for discrimination of 9 domains. "b" is comparison of amino acid sequences of RCCl repeat-containing regions for human ALS2 (hALS2CR6), mouse ALS2 S (mALS2CR6), human (h) RCC I , human (h) RPGR and mouse (m) RPGR. The amino acid residues shown by open frames are the same. Conserved amino acid residues are abundantly contained as well. Positions of the seven blades corresponding to RCC 1 are shown according to the literature3o.
Figure 7 is a chart that compares the wild type human, mouse, and short human variant of the ALS2 proteins and the coding products of the A261de1 (Tunisian) and AGI548de1 (Kuwaiti) mutations.
Detailed Description of the Invention The locus of a 1.7 cM region specified by microsatellite markers D2S116 and D2S2237 of a human second chromosome q 33 region has been mapped3'4. The inventors previously prepared a physical map on the basis of YACBAC/PAC of 3Mb genomic region covering the candidate region in Figure 15'6. Sequences of cDNA clones and EST's have now been analyzed and 42 non-duplicated transcription units including 10 new genes mapped. 411 pairs in of primers were designed depending upon genomic DNA of 14 persons of a family of ALS2 (Fig. 2a) and 6 normal control persons having no kinship with the former was amplified by PCR. Seventy-seven base sequence polymorphs of introns or exons were identified by determining the sequence for all of the PCR products.
Among them, a gene having base deletions related to onset of ALS2 was identified.
The ALS2 gene also includes restriction regions and regulatory regions (promoter/enhancer, suppressor, etc.) which function in expression of protein which is coded thereby. Such restriction and regulation regions are useful for clarifying the functions of the ALS2 gene product as a GEF or a GTPase regulatory factor.
This ALS2 gene may, for example, by isolated by screening a human genome library using pure polynucleotide or oligonucleotide comprising a base sequence of SEQ ID
NO: 1 or a partial sequence thereof as a probe. The resulting genomic gene may be amplified by commonly used genetic amplifying methods such as, for example, a PCR
SUBSTITUTE SHEET (RULE 26) (polymerase chain reaction) method, an NASBN (nucleic acid sequence based amplification) method, a TMA (transcription-mediated amplification) method or an SDA
(strand displacement amplification) method.
A pure polynucleotide (DNA fragments and RNA fragments) may also be prepared from this ALS2 genomic gene, mRNA transcribed by this gene or cDNA synthesized from mRNA. For example, cDNA may be synthesized using poly(A) + RNA extracted from human cells as a template. The human cells may be either those excised from human body by operation, etc. or incubated cells. cDNA may be synthesized by known methods (Mol.
Cell Biol., 2, 161-170, 1982; J. Gene, 25, 263-269, 1983; Gene, 150, 243-250, 1994). One may also synthesize cDNA by an RT-PCR method using an oligonucleotide as a primer and mRNA isolated from human cells as a template. Specifically, the cDNA prepared as such has a base sequence of SEQ ID NO: 1. Those polynucleotides may be used for recombinant expression of a human GTPase regulatory factor.
The oligonucleotides of this invention are DNA fragments or RNA fragments which 1 S hybridize to the above-mentioned ALS2 or the above-mentioned nucleic acids under stringent conditions. For example it is a continuous DNA fragment of 10-100 by in the base sequence of SEQ ID NO: 1. Here, stringent conditions means a condition whereby a specific hybrid formation of target with a probe is made possible by salt concentration, concentration of organic solvent (such as formamide), or temperature condition during hybridization and washing steps. Methods are described in U. S. Patent No.
6,100,037.
One methodology for creating stringent hybridization conditions is [insert B &
K) A primer set of this invention is typically a pair of oligonucleotides for amplification of ALS2 gene or related nucleic acids. Such a primer set may be designed on the basis of the base sequence of SEQ ID NO: 1, synthesized and subjected to purification using known methods. Size (base numbers) of the primer preferably is 15-40 bases or more preferably, 15-30 bases which specificity anneal with a template DNA. However, when LA
(long accurate) PCR is carried out, it is effective to use primers in excess of 30 bases. A pair (two) primers comprising sense strand (5'-terminal side) and antisense strand (3'-terminal side) should not be complementary. In addition, a self complementary sequence is to be avoided in a primer to prevent the formation of a hairpin structure. Further, in order to ensure a stable bond to a template DNA, the GC content should be about SO% and occurrence of GC-SUBSTITUTE SHEET (RULE 26) rich or AT-rich regions in a primer should be avoided. Since an annealing temperature is dependent upon Tm (melting temperature), primers having Tm of 55-65°C
are chosen so as to prepare a PCR product having a high specificity. The final concentration of the primer used in PCR should be about 0.1 to about 1 ~M. It is possible to use commercially available software for designing a primer including the OligoTM software [manufactured by National Bioscience Inc. (U. S. A.)] and GenetyxT"" software [manufactured by Software Development KK (Japan)}.
Mutated ALS2 genes may be obtained by a method where a DNA library prepared from cells of a patient thought to be suffering from ALS2 is screened with a probe which hybridizes to a region containing mutant (e.g. a base deficient site) under a stringent condition. Pure polynucleotide (DNA fragment or RNA fragment) may be obtained from genomic DNA, mRNA or cDNA of an ALS2 mutated gene or a complementary sequence thereof. For example, an ALS2 mutated gene comprises a nucleic acid where the 261 st base a of SEQ ID NO: 1 is deficient. Such a polynucleotide may be used for recombinant 1 S production of ALS2 modified protein or for diagnosis of ALS2.
A primer set for a PCR amplification of ALS2, including various regions having base deficient sites in mutated ALS2 is (for example) a pair of synthetic oligonucleotides comprising base sequences of SEQ ID NO: 6 and NO: 7. This primer set is capable of a PCR amplification of the region (339 bp) including exon 3 and introns before and after that in the ALS2 gene. Another primer set may be composed of synthetic oligonucleotides comprising base sequences of SEQ ID NO: 8 and NO: 9 and is capable of PCR
amplification of exons 2-4 (302 bp) of the ALS2 gene using RNA as a template.
Any PCR
product not cleaved by the restriction enzyme NarI is derived from the normal ALS2 gene but PCR products derived from a mutated ALS2 gene may be cleaved by NarI to give two fragments (Fig. 2c).
A recombinant vector of this invention may be a cloning vector or an expression vector. Vectors will be constructed depending upon the type of the polynucleotide as an insert or upon the object for use. For example, when an ALS2 protein or a modified protein thereof is produced using cDNA or its ORF region as an insert, there may be used an expression vector for an in vitro transcription or an expression suitable for each of prokaryotic cells such as Escherichia coli and Bacillus subtilis and eukaryotic cells such as SUBSTITUTE SHEET (RULE 26) yeast, insect cells and mammalian cells. When a genomic DNA of the ALS2 gene or a mutated gene thereof is used as an insert, it is also possible to use a BAC
(bacterial artificial chromosome) vector or a cosmid vector. Such recombinant vectors are also useful, for example, as probes for diagnosis of chromosome abnormality by hybridization including 5 fluorescent in situ hybridization (FISH). Further, a nucleic acid derived from a normal ALS2 gene may be recombined in a virus vector such as adenovirus or the like and the product may be used for genetic therapy.
In the manufacture of ALS2 peptide (including protein), a transformed cell of this invention may be a prokaryotic cell such as Escherichia coli and Bacillus subtilis or an 10 eukaryotic cell such as from yeast, insects, and mammals. In addition, cells (such as blood stem cells) derived from a patient suffering from ALS2 which are transformed by a virus vector of this invention in which a nucleic acid derived from a normal ALS2 gene is recombined, may be used for a genetic therapy of ALS2. Such transformed cells may be prepared by introducing a recombinant vector into cells by means of known methods such as 15 electroporation, calcium phosphate method, liposome method and DEAE dextran method.
A peptide of this invention may be an expression product of a normal ALS2 gene or an expression product of a mutated ALS2 gene. The normal gene product is a GTPase transcription factor or GEF having an amino acid sequence of SEQ ID NO: 2.
Peptides of this invention are useful as immunogens for the preparation of an antibody, as target molecules for the development of therapeutic agents for ALS2, etc. These peptides may be prepared by methods involving isolating peptides from the cells of healthy persons or patients suffering from ALS2. Methods of chemical synthesis on the basis of a desired amino acid sequence from SEQ ID N0:2 or SEQ ID N0:3, etc. and (preferably) by production and isolation or purification from the above-mentioned transformed cells. Such transformed cells are incubated and isolation and purification are carried out for the culture by, for example, means of treatment with a modifier such as urea or with a surface-active agent, ultrasonic wave treatment, enzymatic digestion, precipitation by salting out or by solvent, dialysis, centrifugal separation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric electrophoresis, ion exchange chromatography, hydrophobic chromatography, affinity chromatography and reversed phase chromatography. Such proteins may include fused proteins with any other protein. For example, fused proteins with glutathione-S-transferase SUBSTITUTE SHEET (RULE 26) (GST) or green fluorescent protein (GFP) may be exemplified. In addition, the protein expressed in cells may be subjected to various kinds of modifications in the cells after being translated. Accordingly, modified proteins are also included in the coverage of the protein of this invention. Examples of the modification after translation as such are elimination of N-terminal methionine, N-terminal acetylation, addition of sugar chain, limited decomposition by intracellular protease, myristoylation, isoprenylation and phosphorylation.
An antibody of this invention is a polyclonal antibody or monoclonal antibody which recognizes a peptide of this invention. Examples include an antibody prepared using a peptide comprising an amino acid sequence of continuous 5 amino acid residues or more of the first to the 46th amino acid sequence in SEQ ID NO: 2 as an antigen and an antibody prepared using a peptide comprising an amino acid sequence of continuous 5 amino acid residues or more of the 47th to the 1657th amino acid sequence in SEQ ID NO: 2 as an antigen. When those two kinds of antibodies are used, it is possible to detect and differentiate normal and A261 del mutant proteins. The antibody of this invention includes all molecules which are able to bind to an epitope of an ALS2 protein or other peptide of this invention, and all of Fab, F(ab')2, Fv fragments, etc. thereof. Such an antibody can be obtained from serum after an animal is immunized using ALS2 derived protein or peptide as an antigen. Alternatively, the above expression vectors for eukaryotic cells may be introduced into muscle or skin of animals by injection or particle gun and then serum is collected therefrom. Examples of animals that may be used are mouse, rat, rabbit, goat, chicken, etc. When B cells collected from the spleen of an immunized animal are fused with myeloma cells to produce a hybridoma, it is possible to produce monoclonal antibodies.
The diagnostic method of this invention is one in which an ALS2 mutated gene or a transcription product of an ALS2 mutated gene is detected whereby the risk of onset of ALS2 may be estimated. Particularly amenable are persons of known ALS2 families although diagnosis is not limited thereto.
Genomic DNA of a person to be diagnosed may be subjected to a PCR
amplification using any of the above-mentioned primer sets or other oligonucleotides of this invention.
The resulting DNA fragment may be treated with one or more restriction enzymes such as NarI, and the person to be diagnosed where the DNA fragment is cleaved into fragments different from cleaving product produced from a person not suffering from ALS2 is SUBSTITUTE SHEET (RULE 26) indicative of a patient suffering from ALS2 or a person with some risk of ALS2 in view of the presence of a mutation in the ALS2 gene.
It is also possible to detect the ALS2 mutated genes by (for example) an allele specific oligonucleotide probe method, an oligonucleotide ligation assay method, a PCR
SSCP method, a PCR-CFLP method, a PCR-PHFA method, an invader method, an RCA
(rolling circle amplification) method and a primer oligo base extension method.
In detecting transcription products of ALS2 mutated genes, diagnosis may be carried out by determining the sequence of mRNA of the person to be diagnosed or cDNA
thereof.
It is also possible to carry out the diagnosis in such a manner that an ALS2 gene of a person to be diagnosed or cDNA thereof is recombined with an expression vector, transfected to cells and the expression product thereof measured.
Expression products of normal and mutant ALS2 genes may be assessed by measurement of molecular weight. For example, the frame shift caused by deletion of one base in normal ALS2 gene whereupon the modified protein is changed to a low-molecular protein (SEQ ID NO: 3) comprising the first to the 46th amino acid residues of SEQ ID NO:
2 and three amino acid residues (Pro-Ser-Glu) newly coded by the frame shift results in a product having a molecular weight easily comparable to naturally occurnng gene products of the normal ALS2 gene. Further, diagnosis may be also carned out by the above antibody provided by this invention in which the ALS2 modified protein reacts with an antibody recognizing (for example) the first to the 46th amino acid sequence in SEQ ID
NO: 2 or a region comprising amino acids 43-49 of SEQ ID N0:3, but does not react with an antibody recognizing the 47th to the 1657th amino acid sequence region in SEQ ID NO: 2.
Antibodies specific for amino acids 476 to 545 of SEQ ID N0:84 as compared to any of the amino acids of SEQ ID N0:2 could be similarly used for diagnosis of the AGI548de1.
Diagnosis using antibodies may, for example, be carried out with an ELIZA
method.
The-mouse ALS2 gene of this invention is a mouse genomic gene isolated as a homolog of the human ALS2 gene and which codes for a mouse ALS2 protein comprising an amino acid sequence of SEQ ID NO: 5. Its cDNA has a base sequence of SEQ ID
NO: 4.
This gene may be used for the preparation of a "knock-out" mouse.
Such a "knock-out" mouse can be prepared by known gene targeting methods (Science, 244: 1288-1292, 1989) or generally according to the following exarriple.
SUBSTITUTE SHEET (RULE 26) First, a DNA fragment of the mouse ALS2 gene including the initiation codon of the gene is modified whereupon a defective DNA fragment which deletes expression of the ALS2 gene is obtained. This defective DNA fragment is used for the preparation of a targeting vector for introduction of the modification into a mouse totipotent cell (ES cell) according to known methods (such as the method described in Science, 244: 1288-1292, 1989). For example, genomic DNA comprising the ALS2 gene is substituted or inserted with a resistant gene to a cytotoxin to prepare a recombinant plasmid DNA
possessing the defective gene having a sequence homologous to the genomic DNA of the ALS2 gene at both terminals (the targeting vector). It is also possible for the resistant gene to be connected to a sequence such as PGKl promoter and PGKl polyadenylation signal for controlling the expression. It is preferred that the genomic DNA site of the ALS2CR6 gene which is substituted with or inserted by resistant gene be a genomic DNA region containing an exon region containing an initiation codon.
There are no particular limitations on such target vectors except that it will have a sequence which is homologous to genomic DNA of the ALS2 gene and a resistance sequence or other sequence useful for cell sorting (such as diphtheria toxin A
gene and thymidine kinase gene of herpes virus). A promoter and enhancer may be appropriately combined and used. The targeting vector is then introduced into an ES
(embryonic stem) cell according to known methods (e.g. Nature, 292: 154-156, 1981). Such methods include electric pulse, a liposome and calcium phosphate. When recombination efficiency of the gene to be introduced is of concern, the electric pulse methods is preferred.
DNA in each of the ES cells into which gene is introduced is extracted and, by means of a southern blot analysis or a PCR assay, cells are selected in which a homologous gene recombination has taken place between the wild type ALS2 gene existing on the chromosome and the introduced defective ALS2 gene fragment resulting in placement of the defective gene fragment in the chromosome.
An ES cell having a defective gene prepared above may be injected into a blastocyst of a wild type animal and chimera-embryos obtained which are transplanted to the uterus of a preliminary parent. Resulting progeny are selected for the ALS2 defective gene and bred.
Selection may be carned out by checking the difference in the color of hair or by extraction of DNA from a part of the body (such as the tail end) followed by conducting a southern blot SUBSTITUTE SHEET (RULE 26) analysis, a PCR assay after extraction of DNA, etc. As to the offspring obtained by a crossbreeding of animal of a wild type with a chimera animal where the ALS2 defective gene is in the generative cells, a southern blot analysis, a PCR assay or the like may be carned out using the DNA extracted from a part of the body (such as the tail end) as a material to identify a heterozygote into which the ALS2 defective gene is introduced. A
heterozygote possessing the ALS2 defective gene which is stable in all generative cells and somatic cells may be bred to produce progeny in which the ALS2 gene is completely knocked-out".
An animal prepared as such may be used for analysis of function of ALS2 gene in onset of ALS2 and for screening of therapeutic drugs or development of therapeutic methods as an ALS2 model animal.
Methods and results of procedures carned out for cloning of the ALS2 gene and for functional analysis thereof are shown.
1. Methods 1-1. ALS2 family Sixteen cases including 8 individuals suffering from the disease obtained from a Tunisian consanginous ALS2 family (literature 2) were analyzed. The characteristic of ALS2 is a progressive convulsion of muscles of the limbs and the face accompanied by distal myoatrophy of the hand and the foot. Age of onset is between 3 and 10 years age (literature 2). According to biopsy of nerves and muscles and also to electromyography test, there was confirmed deletion of distal motor neuron (literature 2). When a gene type of the ' polymorphic DNA markers was analyzed together with clinical test data, ALS2 was clearly an autosomal recessive inheritance.
1-2. Transcription map Genome Data Base (GDB) (http://www.gdbwww.gdb.org) and UniGene (http://www.ncbi.nJm.nih.gov) of the Biotechnology Information Center (NCBI) which were open to the public for discriminating the sequence of transcribed DNA mapped within an objective region were retrieved. Sequence of genomic DNA. overlapped with the objective region of ALS2 was retrieved from the "nr" or "htgs" data base of GenBank and utilized as the object for the test when a BLAST retrieval to the dbEST data base is conducted. In order to isolate the transcript of a full length, there were carned out RT-PCR, 5'-RACE and cDNA
SUBSTITUTE SHEET (RULE 26) library screening. In addition, EST clone was purchased from Research Genetics and sequencing for DNA was carned out for measuring the insertion of the whole clone.
Sequence of double stranded DNA was determined by conducting a dideoxy sequencing using a BigDye Terminator Cycle Sequencing Kit (ABI) and an AB 1377DNA
sequences.
5 All sequences of EST data, PCR products and DNA obtained from cDNA clone were determined and an estimated independent transcription unit was established.
Then each unit was mapped on a physical map by a PCR method.
1-3. Identification of exon In order to determine the constitution of intron and exon of the transcription DNA, 10 genomic DNA sequence data open to the public in GenBank data base was compared with the sequence of cDNA using a Sequences Version 3.0 (Gene Codes Corporation) program according to the descriptions of BLAST (literature 28) and literatures (5 and 6).
1-4. PCR
Exon and intron/exon boundaries were subjected to a PCR amplification. ExTaq 15 polyrnerase (Takara) was used and a cycle of 95°C for 15 seconds, 60°C for 30 seconds and 72°C for 30 seconds was repeated for 35 times whereby about SO mg of genomic DNA were amplified by a PCR. In order to detect the deficient form of the transcription DNA, an RT-PCR was carried out. Total RNA from lymphocytes of four patients of a family of ALS2 and two carriers was isolated. Total RNA extracted from a healthy human brain was 20 purchased from Clontech. An RT-PCR was carried out using a Superscript pre-amplification system (Gibco-BRL) according to the protocol of the manufacturer. The oligonucleotide primer for such a PCR was designed using Primer 3.0 (http://www-genome.wi.mit.edu). Table 1 lists the primers used for amplification of ALS2 (ALS2CR6).
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SUBSTITUTE SHEET (RULE 26) 1-5. Analysis of mutation In order to detect the mutation of the DNA sequence at the exon or the intron/exon boundary, a DNA sequence of a PCR product of exon was determined. The DNA
sequence of a PCR product of exon was analyzed using the same oligonucleotide as a primer. The S sequence in the data base open to the public was compared with the DNA
sequence obtained from patients, carriers and healthy persons and changes in the nucleotide were discriminated.
[0044]
It was also confirmed that a new NarI site was formed (A261 del) after the treatment with NarI by means of an RT-PCR amplification of exons 2-4 or a PCR
amplification of exon 3. As to the primers for exon 3-PCR, there were used 5' CCTAGTCATCCATGTGCTGG-3' (SEQ ID NO: 6) and 5' TCCCATACCTGACCTTCCAC-3' (SEQ ID NO: 7). As to the primers for the RT-PCR of exons 2-4, there were used 5'-CTTGATAGACTTTCTGTAAAGAAG-3' (SEQ ID NO: 8) and 5'-GGCTACTTGGACAAATCTCCACTG-3' (SEQ ID NO: 9). Decomposed product with NarI was separated by 1.5% agarose gel.
1-6. Northern blot analysis Northern (MTN) blot (Clontech) of many human adult tissues was hybridized with exon 4 labelled with 32P-dCTP of ALS2CR6 or human (3-actin cDNA in a Perfect Hyb hybridizing solution (Toyobo). The membrane was washed with 0.1 x SSC
containing 1%
of SDS and subjected to an X-ray film (Bio-MAX, Kodak).
1-7. mRNA in situ hybridization Antisense and sense cRNA probes were prepared from two mouse cDNA clones m2-as and m2-s. Those mouse cDNA clones covered a part of mouse mALS2CR6 cDNA
(from the 1732nd to the 2685th bases of SEQ ID NO: 4; 954 bp) and inserted into pCR2.1 (Invitrogen) in an opposite direction. The probes were prepared according to the protocol of the manufacturer (Roche Molecular Biochemicals) by an in vitro transcription reaction where digoxigenin-labelled UTP and T7 polymerase were mixed. Preparation of the sample and method for the in situ hybridization were in accordance with the literature (29).
1-8. Retrieval of the data base Each of DNA and amino acid sequences was compared with the data base of sequences of nucleotide and protein which were not overlapped each other using BLASTN
SUBSTITUTE SHEET (RULE 26) and BLASTP. Domain and motive of protein were identified by MOTIF servers of Genome Net Japan (http://www.~enome.ad.jp), search launcher of BCM
(http://www.hgse.hem.tmc.edu/Search.launcher) and CD search of NCBI
(http://www.hcbi.nlm.nih.~ov).
S 2. Results The inventors have prepared a physical map on the basis of YAC/BAC/PAC of genomic region of 3 Mb covering a complete candidate region to ALS2 (literatures 5 and 6).
Sequences of EST and cDNA clone were analyzed within a broad area and, at the same time, this physical map was used for the mapping of 43 independent transcription units including previously analyzed 18 genes (KIAA0005, CLKl, PP1L3, ORC2L, NDUFB3, CFLAR, CASP10, CASPB, FZD7, NOPS, UBL1, BMPR2, FLJ10881, LOC57404, AIP-1, CD28, CTLA4 and AILIM) and new 10 full-length transcription products (ALS2CR1, ALS2CR2, ALS2CR3, ALS2CR4, ALS2CR5/MPP4, ALS2CR6, ALS2CR7, ALS2CR8, ALS2CR9 and ALS2CR12). Those genetic sequences were present in the locus of ALS2 (Fig.
1 ).
[0045]
Juvenile ALS2 is rare and has a sign that, in teens and twenties, muscular convulsion of limbs, face and throat gradually expresses. Since ALS2 is recessively hereditary, it is predicted that this ALS2 disease may take place by a loss of a functional mutation. . Big deletion or translocation in the ALS2 locus was investigated by a mapping of STS/EST
content on the basis of a PCR and a southern blot analysis but that was not detected. After that, small deletion or base substitution in exon or intron-exon boundary was investigated.
In order to detect those mutations, each gene was analyzed and an intron/exon boundary thereof was determined. Until now, 395 exons have been identified from 42 genes. In order to amplify exon and flanking sequence thereof including consensus sequence to splicing donor and acceptor, 411 primers in total were designed and those primers were used to amplify the genomic DNA of 10 normal control persons who were not related to 14 persons of the ALS2 family (Fig. 2a) by PCR. Sequence of each of those PCR products was determined whereby 77 sequence polymorphs in total of intron or exon were identified.
Among those 77 polymorphs, 8 mutations contained in 4 different genes were related to ALS2 (Table 2).
SUBSTITUTE SHEET (RULE 26) Table 2 Gene Region Normal ALS2 NOPS intron 2 tatctc(T~9aattct NOPS intron 6 gttttg(TTG 2ttttta ~ (TTG)3 ALS2CR6 intron 2 ggtaaAtcattt ALS2CR6 exon 3 gcaggcAgccctc -~ A261 deletion*

ALS2CR8 intron 6 gtcagtAttataa ALS2CR9 exon 4 ctccagCatggac ~ T (3rd codon) ALS2CR9 intron 7 ttgggaTtttttt ALS2CR9 intron 8 aaaataCggatat .-~ T

Among those sequence mutations, one nucleotide deletion (A261 del) noted in exon 3 of ALS2CR6 broke the reading frame and it is suggested that such a mutation mutates the protein. All of the suspicious hetero-conjugative carriers show a duplicated sequence pattern starting from the first nucleotide after the deficient part (Fig. 2b). This deletion clearly moves together with an ALS2 expression type (Fig. 2c) and is not noted in 533 normal control individuals of various races (data not shown). In other mutations, one base substitution from C to T in exon 4 of ALS2CR9 gene is included (C873T).
However, this mutation corresponds to the third codon and, therefore, it does not change the amino acid residue. In order to detect a splicing error which is made latent or manifest by other sequence mutation, an RT-PCR was carned out using total RNA extracted from lymphocytes of patients and healthy control persons but no sequence mutation of mRNA
was detected (data not shown). Accordingly, the mutation related to ALS2 of intron or exon region does not cause a splicing error. From those results, it has been confirmed that deletion of one base in exon 3 of ALS2CR6 (A261 del; Table 1 ) is mutation concerning ALS2.
ALS2CR6 gene contains 33 introns and 34 exons and is present in a genomic DNA
of 80.3 kb adjacent to a polymorphic DNA marker D2S2309 (Fig. 1).
Transcription polarity of the ALS2CR6 gene is in the direction of central body from telomere. An transcription product (mRNA) has a full length of 6394 by (SEQ ID NO: 1) having a single open reading frame (ORF) with a length of 4974 nucleotides (124-5,097 nt) and codes for a SUBSTITUTE SHEET (RULE 26) protein of 184 KDa comprising 1,657 amino acid residues. Polyadenylated estimated signal (AATAAA: 6,375-6,380 nt) and poly(A) region are clear. A short ALS2CR6 transcription product in a full length of 2,651 by having 1,191 by ORF coding for a 396 amino acid sequence was identified as well. This short variant splices a 5'-donor site after exon 4 and, S as a result, a stop codon is formed after 25 amino acid residues of intron 4. Being correspondent to those results, 2 transcription products of about 6.5 kb and 2.6 kb were identified in many adult human tissues by a northern blot analysis (Fig. 3a).
Except the liver where short transcription products are mostly expressed, both transcription products showed the similar expression pattern. It has been confirmed that a big transcription product of 6.5 10 kb is expressed in a slightly higher level than a transcription product of 2.6 kb and is most abundantly expressed in the cerebellum. This gene has been also confirmed to be expressed in cells of ALS2 patients (Fig. 3b).
Further, a mouse homolog of ALS2CR6 was isolated and named mALS2CR6. A
transcription product of mALS2CR6 is in a full length of 6,349 by (SEQ ID NO:
4) having 15 one ORF of 4,956 by (124-5,076 nt) and codes for a protein of 183 kDa comprising 1651 amino acids (SEQ ID NO: 5). The ORF as a whole is well reserved in a DNA level (87%
same) and a protein level (91 % same; 94% similar; Fig. 4) between human being and mouse and it is suggested that ALS2CR6 gene is a gene which is well reserved in mammals.
In order to check the localization property of expression of mALS2CR6 transcription 20 product in the brain and the spinal cord of mouse, an in situ hybridization using riboprobe corresponding to a part of mALS2CR6 cDNA was carned out. The result was that, as shown in Fig. 5, the mALS2CR6 transcription products were expressed in various levels in nerve cells from the brain to the spinal cord, especially in neurons of hippocampal and dentate gyrus, cerebellar Purkinje cells, neurons of cerebral cortex and spinal cinerea 25 including anterior horn cells. In addition, a significant expression was noted in neurons of olfactory bulb, basal nucleus and cranial nerve nucleus as well.
Human ALS2CR6 protein showed many interesting properties (Fig. 6a). The first property is present in a region of A-terminal side of ALS2CR6 and it showed a high homology to RCC 1 (regulatory factor for concentrating the chromosome;
literature 7) and RPGR (GTPase for pigmentary retinitis; literature 8)(Fig. 8). RCC1 and RPGR
protein acts as a guanine nucleotide exchange factor (GEF) for GTPase like Ran. The second property is SUBSTITUTE SHEET (RULE 26) that ALS2CR6 has a Dbl-homologous (DH) domain and a pleckstrin-homologous (PH) domain and both domains are typical domains noted in RhoGEF protein (literatures 9 and 10). In addition, VPS9 domain is noted in a C-terminal region as well. VPS9 domain is noted in many GEF including Vps9 (literature 11 ) and Rabex-S (literature 12) and each is said to mediate the selection of vacuole protein and the phagocytic transportation. Two MORN motives comprising 14 amino acids (literature 13) were noted as well.
According to the recent study for junctophilin containing an MORN motive, this motive is shown to contribute in bonding of plasma membrane (literature 13). It has been known that GEF is related to a GDP bonding form of GTPase and promotes the dissociation of GDP
and bonding of GTP whereby GTPase is activated. Since it has been known (literatures 18 and 19) that GEF plays an important role in many signal transmission cascades (literature 14), neuron formation (literature 15), membrane transportation (literature 16) and formation of actin cell skeleton (literature 17), it is likely that ALS2CR6 acts as a regulatory factor/activator of Ran-related GTPase, regulates the formation of membrane, and acts in a (membrane) transportation of cells including neurons.
According to an RT-PCR analysis, a transcription product of mutated ALS2CR6 gene is transcribed from chromosomes of the patient (Fig. 2c) and produces a modified protein comprising 49 amino acids having three new residues (Pro-Ser-Glu) at C-terminal (Fig. 6a). Since this modified protein has no functional domain corresponding to ALS2CR6 protein, it seems to make the inherent function lost. Accordingly, the A261de1 mutation noted in this ALS2CR6 is related to the fact that ALS2 is recessively hereditary.
A recent finding that ALS is related to defect for the transportation of axon and the formation of cell skeleton (literatures 20, 21 and 22) induces a hypothesis that ALS2CR6 gene corresponds to ALS2 and that ALS2 is generated by the defect of membrane structure due to lacking in a regulatory function of membrane structure Ran-related GTPase.
ALS2CR6 gene is the second ALS gene succeeding to the determination of role of copper-zinc superoxide desmutase (SDS-1) in~ ALS. Mutation of SOD-1 is related to the form of tardive autosomal dominance (literature 23).
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of skill in the art in light of the teachings of this invention that changes and SUBSTITUTE SHEET (RULE 26) modification may be made thereto without departing from the spirit or scope of the appended claims. All patents, patent applications and publications referred to herein are hereby incorporated by reference.
S
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SUBSTITUTE SHEET (RULE 26) SEQUENCE LISTING
<110> The University of British Columbia; and Japan Science and Technology Corporation <120> ALS2 Gene and Amyotrophic Lateral Sclerosis Type 2 <130> 80021-346 <140>
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<150> US 60/267,723 <151> 2001-02-12 <150> JP 2001-116973 <151> 2001-04-16 <150> US 60/318,352 <151> 2001-09-12 <160> 84 <170> PatentIn Ver. 2.0 <210> 1 <211> 6394 <212> DNA
<213> Homo Sapiens <220>
<221> CDS
<222> (124)..(5097) <400> 1 ggacccactg ggttgccaag ctcgcgccgg atgcggagcg cggtgctgcc ggtggagctt 60 caggtcttga tagactttct gtaaagaagg aatgatttgg tgatggagtg ttcccactga 120 ccg atg gac tca aag aag aga agc tca aca gag gca gaa gga tcc aag 168 Met Asp Ser Lys Lys Arg Ser Ser Thr Glu Ala Glu Gly Ser Lys gaa aga ggc ctg gtc cat atc tgg cag gca gga tcc ttt ccc ata aca 216 Glu Arg Gly Leu Val His Ile Trp Gln Ala Gly Ser Phe Pro Ile Thr cca gag aga ttg cca ggc tgg gga gga aag act gtt ttg cag gca gcc 264 Pro Glu Arg Leu Pro Gly Trp Gly Gly Lys Thr Val Leu Gln Ala Ala ctc gga gtg aaa cat gga gtt ctt ctg act gaa gat ggt gag gtc tac 312 Leu Gly Val Lys His Gly Val Leu Leu Thr Glu Asp Gly Glu Val Tyr agc ttt ggg act ctt ccc tgg aga agt gga cca gtg gag att tgt cca 360 Ser Phe Gly Thr Leu Pro Trp Arg Ser Gly Pro Val Glu Ile Cys Pro agt agc ccc att cta gaa aat gcc ctg gtt ggg caa tat gtt att act 408 Ser Ser Pro Ile Leu Glu Asn Ala Leu Val Gly Gln Tyr Val Ile Thr SUBSTITUTE SHEET (RULE 26) gtg gca aca gga agc ttc cat agt gga gca gtg aca gac aat ggt gtc 456 Val Ala Thr Gly Ser Phe His Ser Gly Ala Val Thr Asp Asn Gly Val gcg tac atg tgg gga gag aat tct get ggc cag tgt gca gta gcc aac 504 Ala Tyr Met Trp Gly Glu Asn Ser Ala Gly Gln Cys Ala Val Ala Asn cag cag tat gtg ccg gaa cca aat cct gtc agc att get gat tct gag 552 Gln Gln Tyr Val Pro Glu Pro Asn Pro Val Ser Ile Ala Asp Ser Glu gcc agc cct ttg tta gca gtc agg att tta cag ttg gcg tgt ggc gag 600 Ala Ser Pro Leu Leu Ala Val Arg Ile Leu Gln Leu Ala Cys Gly Glu gag cac act ctg gca ttg tca ata agc aga gag att tgg gca tgg ggt 648 Glu His Thr Leu Ala Leu Ser Ile Ser Arg Glu Ile Trp Ala Trp Gly acc ggt tgt cag ttg ggt ctc att acc act gcc ttc cca gtg aca aag 696 Thr Gly Cys Gln Leu Gly Leu Ile Thr Thr Ala Phe Pro Val Thr Lys ccg caa aag gta gaa cat ctt get ggg cga gtg gtg ctt caa gtt gcc 744 Pro Gln Lys Val Glu His Leu Ala Gly Arg Val Val Leu Gln Val Ala tgt ggt get ttc cac agc tta gcc ctt gta caa tgc ctc cct tcc cag 792 Cys Gly Ala Phe His Ser Leu Ala Leu Val Gln Cys Leu Pro Ser Gln gat ctg aag cca gtc cca gaa cga tgc aac cag tgc agc cag ctc ttg 840 Asp Leu Lys Pro Val Pro Glu Arg Cys Asn Gln Cys Ser Gln Leu Leu att act atg act gac aaa gaa gac cat gtg att ata tca gac.agt cat 888 Ile Thr Met Thr Asp Lys Glu Asp His Val Ile Ile Ser Asp Ser His tgt tgc cca tta ggt gtg aca ctg aca gaa tct cag gca gaa aac cat 936 Cys Cys Pro Leu Gly Val Thr Leu Thr Glu Ser Gln Ala Glu Asn His gcc agc act get ctc agc ccc tcc act gaa acc ctt gac agg cag gaa 984 Ala Ser Thr Ala Leu Ser Pro Ser Thr Glu Thr Leu Asp Arg Gln Glu gaa gta ttt gag aac act ctt gta gca aat gat cag tct gtt get act 1032 Glu Val Phe Glu Asn Thr Leu Val Ala Asn Asp Gln Ser Val Ala Thr gaa ctg aat gca gta agt get cag atc aca agc agc gat gcc atg tcc 1080 Glu Leu Asn Ala Val Ser Ala Gln Ile Thr Ser Ser Asp Ala Met Ser tct caa caa aat gtc atg gga aca act gaa att tcc tct gcc aga aac 1128 Ser Gln Gln Asn Val Met Gly Thr Thr Glu Ile Ser Ser Ala Arg Asn ata cca tca tac cct gac acc caa gca gtc aat gaa tac cta cgg aaa 1176 SUBSTITUTE SHEET (RULE 26) Ile Pro Ser Tyr Pro Asp Thr Gln Ala Val Asn Glu Tyr Leu Arg Lys ctg tca gat cat tca gta aga gag gac tca gag cat ggt gaa aag cca 1224 Leu Ser Asp His Ser Val Arg Glu Asp Ser Glu His Gly Glu Lys Pro atg cca tct cag cct ctt tta gaa gaa gca att cct aat ctc cac agc 1272 Met Pro Ser Gln Pro Leu Leu Glu Glu Ala Ile Pro Asn Leu His Ser ccg cct acc aca agc acc tca gcc cta aac agc ctg gtg gtc tct tgt 1320 Pro Pro Thr Thr Ser Thr Ser Ala Leu Asn Ser Leu Val Val Ser Cys gca tct get gtt ggt gtg aga gtg get get act tat gaa get ggt gcc 1368 Ala Ser Ala Val Gly Val Arg Val Ala Ala Thr Tyr Glu Ala Gly Ala ttg tca ctg aag aaa gtt atg aac ttt tat agt aca acc cct tgt gaa 1416 Leu Ser Leu Lys Lys Val Met Asn Phe Tyr Ser Thr Thr Pro Cys Glu act gga get cag gca ggc agt agt gcc att ggc ccc gaa ggt ttg aaa 1464 Thr Gly Ala Gln Ala Gly Ser Ser Ala Ile Gly Pro Glu Gly Leu Lys gat agc agg gaa gaa cag gtt aaa cag gaa tca atg caa gga aag aaa 1512 Asp Ser Arg Glu Glu Gln Val Lys Gln Glu Ser Met Gln Gly Lys Lys agt tca agt ctt gtg gat atc aga gaa gaa gaa aca gag gga ggc agt 1560 Ser Ser Ser Leu Val Asp Ile Arg Glu Glu Glu Thr Glu Gly Gly Ser cga aga ctc tcc ctc cct gga ttg ttg tca caa gtt tcc ccc agg ctc 1608 Arg Arg Leu Ser Leu Pro Gly Leu Leu Ser Gln Val Ser Pro Arg Leu tta aga aag get gca cgg gtg aaa acg agg aca gtg gtt ctg acc ccc 1656 Leu Arg Lys Ala Ala Arg Val Lys Thr Arg Thr Val Val Leu Thr Pro aca tac agt gga gaa gca gat gcg ctc ctg cct tct ctg aga aca gaa 1704 Thr Tyr Ser Gly Glu Ala Asp Ala Leu Leu Pro Ser Leu Arg Thr Glu gtg tgg acc tgg ggg aaa ggg aag gaa ggg cag ctg ggg cac ggc gat 1752 Val Trp Thr Trp Gly Lys Gly Lys Glu Gly Gln Leu Gly His Gly Asp gtt ctg cct agg ctt caa ccg ttg tgt gta aaa tgt ctg gat ggc aaa 1800 Val Leu Pro Arg Leu Gln Pro Leu Cys Val Lys Cys Leu Asp Gly Lys gaa gta atc cat ctg gag gca ggt ggt tac cat tct ctt gca ctt act 1848 Glu Val Ile His Leu Glu Ala Gly Gly Tyr His Ser Leu Ala Leu Thr gcg aaa tcc cag gtt tac tca tgg ggt agc aat acc ttt ggt caa ctt 1896 Ala Lys Ser Gln Val Tyr Ser Trp Gly Ser Asn Thr Phe Gly Gln Leu SUBSTITUTE SHEET (RULE 26) ggg cat tcc gat ttt cca aca aca gtt cct cgt ctt gca aag ata agc 1944 Gly His Ser Asp Phe Pro Thr Thr Val Pro Arg Leu Ala Lys Ile Ser agt gaa aat gga gtc tgg agc ata get gca ggc agg gat tat tcc ctg 1992 Ser Glu Asn Gly Val Trp Ser Ile Ala Ala Gly Arg Asp Tyr Ser Leu ttt tta gtg gat aca gaa gac ttc cag cct ggg tta tat tac agt ggc 2040 Phe Leu Val Asp Thr Glu Asp Phe Gln Pro Gly Leu Tyr Tyr Ser Gly cga cag gac cct aca gaa ggt gac aac ctt cca gag aat cac agt ggt 2088 Arg Gln Asp Pro Thr Glu Gly Asp Asn Leu Pro Glu Asn His Ser Gly tct aag act cca gta ctt ctc tcc tgt agt aag ctt gga tat ata agc 2136 Ser Lys Thr Pro Val Leu Leu Ser Cys Ser Lys Leu Gly Tyr Ile Ser aga gtg aca gca gga aaa gat agc tat tta gcc ttg gtg gat aaa aac 2184 Arg Val Thr Ala Gly Lys Asp Ser Tyr Leu Ala Leu Val Asp Lys Asn att atg ggg tat att gcc agt ctc cac gag tta get act aca gaa aga 2232 Ile Met Gly Tyr Ile Ala Ser Leu His Glu Leu Ala Thr Thr Glu Arg cga ttc tat tca aaa cta agt gat atc aaa tct cag att ctc agg cct 2280 Arg Phe Tyr Ser Lys Leu Ser Asp Ile Lys Ser Gln Ile Leu Arg Pro ctt ctc agt tta gaa aat ttg ggc act aca act aca gtc cag ctg ttg 2328 Leu Leu Ser Leu Glu Asn Leu Gly Thr Thr Thr Thr Val Gln Leu Leu cag gag gtg get agc cga ttc agc aag ctg tgt tac ctc att ggt cag 2376 Gln Glu Val Ala Ser Arg Phe Ser Lys Leu Cys Tyr Leu Ile Gly Gln cat gga gcc tca ttg agc agc ttc ctt cat ggg gta aag gaa gcc agg 2424 His Gly Ala Ser Leu Ser Ser Phe Leu His Gly Val Lys Glu Ala Arg agt ttg gtc atc ctg aag cat tca agt ctc ttc ttg gat agt tat aca 2472 Ser Leu Val Ile Leu Lys His Ser Ser Leu Phe Leu Asp Ser Tyr Thr gag tat tgc aca tct att aca aat ttc ctg gtt atg gga gga ttc cag 2520 Glu Tyr Cys Thr Ser Ile Thr Asn Phe Leu Val Met Gly Gly Phe Gln ctt ctt get aag cct gcc att gat ttc cta aat aaa aac caa gag ctg 2568 Leu Leu Ala Lys Pro Ala Ile Asp Phe Leu Asn Lys Asn Gln Glu Leu ttg caa gat ttg tca gaa gtg aat gac gaa aac act cag ttg atg gaa 2616 Leu Gln Asp Leu Ser Glu Val Asn Asp Glu Asn Thr Gln Leu Met Glu ata ctg aat act ttg ttt ttc ttg cca atc aga cga ctt cat aat tac 2664 SUBSTITUTE SHEET (RULE 26) Ile Leu Asn Thr Leu Phe Phe Leu Pro Ile Arg Arg Leu His Asn Tyr gca aaa gtt ttg cta aag ctt get act tgt ttt gaa gtg gca tct cca 2712 Ala Lys Val Leu Leu Lys Leu Ala Thr Cys Phe Glu Val Ala Ser Pro gaa tat cag aaa ctg cag gat tcc agt tct tgt tat gag tgt ctt get 2760 Glu Tyr Gln Lys Leu Gln Asp Ser Ser Ser Cys Tyr Glu Cys Leu Ala ctc cat ctc ggc agg aaa agg aag gaa gca gaa tac aca ctg ggc ttc 2808 Leu His Leu Gly Arg Lys Arg Lys Glu Ala Glu Tyr Thr Leu Gly Phe tgg aag acc ttc ccc gga aaa atg acg gat tcc ttg agg aag cca gag 2856 Trp Lys Thr Phe Pro Gly Lys Met Thr Asp Ser Leu Arg Lys Pro Glu cgt cga ctg ctg tgt gag agt agt aac cga gcc ctg tct ctg cag cat 2904 Arg Arg Leu Leu Cys Glu Ser Ser Asn Arg Ala Leu Ser Leu Gln His get ggg agg ttt tcc gtg aat tgg ttc att ctc ttt aat gat gcc ctg 2952 Ala Gly Arg Phe Ser Val Asn Trp Phe Ile Leu Phe Asn Asp Ala Leu gtc cat gcc cag ttc tcc acg cac cat gtt ttc cct ctg gcc acg ctg 3000 Val His Ala Gln Phe Ser Thr His His Val Phe Pro Leu Ala Thr Leu tgg gca gag cca ctg tct gaa gaa get ggt ggt gtg aat ggc tta aag 3048 Trp Ala Glu Pro Leu Ser Glu Glu Ala Gly Gly Val Asn Gly Leu Lys ata act aca cct gag gag cag ttc act ctc att tca tct aca ccc cag 3096 Ile Thr Thr Pro Glu Glu Gln Phe Thr Leu Ile Ser Ser Thr Pro Gln gaa aag aca aag tgg cta cga get ata agc caa gcc gta gat cag get 3144 Glu Lys Thr Lys Trp Leu Arg Ala Ile Ser Gln Ala Val Asp Gln Ala ttg aga ggg atg tct gat ctc ccc cct tat gga agt ggt agc agt gtt 3192 Leu Arg Gly Met Ser Asp Leu Pro Pro Tyr Gly Ser Gly Ser Ser Val cag aga cag gaa cca ccc att tca cgc agt gcc aaa tat act ttc tac 3240 Gln Arg Gln Glu Pro Pro Ile Ser Arg Ser Ala Lys Tyr Thr Phe Tyr aag gat cct cgc cta aag gat gcc acc tat gat gga cgc tgg ctt tca 3288 Lys Asp Pro Arg Leu Lys Asp Ala Thr Tyr Asp Gly Arg Trp Leu Ser ggg aag cct cat ggc aga ggg gtt ttg aag tgg cct gat gga aag atg 3336 Gly Lys Pro His Gly Arg Gly Val Leu Lys Trp Pro Asp Gly Lys Met tat tct ggc atg ttc agg aat ggc ttg gaa gat ggg tat gga gaa tac 3384 Tyr Ser Gly Met Phe Arg Asn Gly Leu Glu Asp Gly Tyr Gly Glu Tyr SUBSTITUTE SHEET (RULE 26) aga atc cca aac aag gca atg aac aaa gaa gac cat tat gtg ggc cat 3432 Arg Ile Pro Asn Lys Ala Met Asn Lys Glu Asp His Tyr Val Gly His tgg aaa gaa gga aaa atg tgc ggt caa gga gtc tac agc tat get tct 3480 Trp Lys Glu Gly Lys Met Cys Gly Gln Gly Val Tyr Ser Tyr Ala Ser ggt gaa gta ttt gag ggc tgt ttt caa gat aat atg cgt cat ggt cat 3528 Gly Glu Val Phe Glu Gly Cys Phe Gln Asp Asn Met Arg His Gly His ggt ctt cta cga agt ggg aaa ttg acg tcc tct tct cct agt atg ttc 3576 Gly Leu Leu Arg Ser Gly Lys Leu Thr Ser Ser Ser Pro Ser Met Phe att ggc cag tgg gta atg gat aag aaa gca gga tat ggt gtc ttt gat 3624 Ile Gly Gln Trp Val Met Asp Lys Lys Ala Gly Tyr Gly Val Phe Asp gat atc act agg ggg gaa aag tat atg gga atg tgg caa gat gat gtg 3672 Asp Ile Thr Arg Gly Glu Lys Tyr Met Gly Met Trp Gln Asp Asp Val tgt caa ggg aat ggt gtg gtg gtt acc cag ttt gga tta tac tac gag 3720 Cys Gln Gly Asn Gly Val Val Val Thr Gln Phe Gly Leu Tyr Tyr Glu ggc aac ttt cac ctt aat aaa atg atg gga aat ggg gtt ttg ctt tcc 3768 Gly Asn Phe His Leu Asn Lys Met Met Gly Asn Gly Val Leu Leu Ser gaa gat gat act atc tat gaa gga gaa ttt tca gat gac tgg act ctt 3816 Glu Asp Asp Thr Ile Tyr Glu Gly Glu Phe Ser Asp Asp Trp Thr Leu agt gga aag gga aca ctg act atg cca aat gga gac tac att gaa ggt 3864 Ser Gly Lys Gly Thr Leu Thr Met Pro Asn Gly Asp Tyr Ile Glu Gly tat ttt agt gga gaa tgg gga tct ggg ata aaa atc act gga acc tac 3912 Tyr Phe Ser Gly Glu Trp Gly Ser Gly Ile Lys Ile Thr Gly Thr Tyr ttc aaa cct agt cta tat gag agt gat aaa gac aga cct aaa gtt ttc 3960 Phe Lys Pro Ser Leu Tyr Glu Ser Asp Lys Asp Arg Pro Lys Val Phe agg aag cta gga aac ctg gca gtg cca get gat gag aag tgg aaa gcg 4008 Arg Lys Leu Gly Asn Leu Ala Val Pro Ala Asp Glu Lys Trp Lys Ala gtg ttt gac gaa tgt tgg cgc caa ctg ggc tgt gag ggc cca ggc caa 4056 Val Phe Asp Glu Cys Trp Arg Gln Leu Gly Cys Glu Gly Pro Gly Gln ggg gaa gtt tgg aaa gca tgg gac aat att get gtg gcc ttg acc acc 4104 Gly Glu Val Trp Lys Ala Trp Asp Asn Ile Ala Val Ala Leu Thr Thr agt cgg cgc cag cac aga gac agt cca gaa ata ttg agt cgt tca cag 4152 SUBSTITUTE SHEET (RULE 26) Ser Arg Arg Gln His Arg Asp Ser Pro Glu Lle Leu Ser Arg Ser Gln act cag aca cta gag agt ttg gaa ttc att cca cag cat gtt ggt gcc 4200 Thr Gln Thr Leu Glu Ser Leu Glu Phe Ile Pro Gln His Val Gly Ala ttc tct gtg gag aaa tat gat gac atc agg aaa tat tta ata aag gcc 4248 Phe Ser Val Glu Lys Tyr Asp Asp Ile Arg Lys Tyr Leu Ile Lys Ala tgt gac act cct ctg cac ccc ctg ggc agg ctt gtg gag aca ctg gtt 4296 Cys Asp Thr Pro Leu His Pro Leu Gly Arg Leu Val Glu Thr Leu Val gca gtg tat aga atg aca tac gtg ggc gta gga gcc aac cgc agg tta 4344 Ala Val Tyr Arg Met Thr Tyr Val Gly Val Gly Ala Asn Arg Arg Leu ttg cag gag get gta aag gag att aag tcc tat ctt aag cga att ttc 4392 Leu Gln Glu Ala Val Lys Glu Ile Lys Ser Tyr Leu Lys Arg Ile Phe cag ctg gtg agg ttc tta ttt cct gag ctg cct gaa gaa ggc agc aca 4440 Gln Leu Val Arg Phe Leu Phe Pro Glu Leu Pro Glu Glu Gly Ser Thr att cct ctc tct get cct ctg cca acc gaa agg aag tct ttt tgc act 4488 Ile Pro Leu Ser Ala Pro Leu Pro Thr Glu Arg Lys Ser Phe Cys Thr ggg aag tca gat tcc cga tct gaa tca cca gag cca ggt tat gta gta 4536 Gly Lys Ser Asp Ser Arg Ser Glu Ser Pro Glu Pro Gly Tyr Val Val acg agt tct gga tta ttg ctt cct gtg ctg cta cct cgg ctc tac cca 4584 Thr Ser Ser Gly Leu Leu Leu Pro Val Leu Leu Pro Arg Leu Tyr Pro ccg ctg ttt atg ctt tat get ttg gat aat gat cgc gag gaa gac att 4632 Pro Leu Phe Met Leu Tyr Ala Leu Asp Asn Asp Arg Glu Glu Asp Ile tac tgg gaa tgt gtc ctt cga cta aat aag cag cca gat att get ctc 4680 Tyr Trp Glu Cys Val Leu Arg Leu Asn Lys Gln Pro Asp Ile Ala Leu ctg ggc ttt ctt ggg gtg cag agg aaa ttt tgg cca gca acc ttg tca 4728 Leu Gly Phe Leu Gly Val Gln Arg Lys Phe Trp Pro Ala Thr Leu Ser atc ctt gga gag agt aaa aag gtt ttg cca acc acg aaa gat get tgt 4776 Ile Leu Gly Glu Ser Lys Lys Val Leu Pro Thr Thr Lys Asp Ala Cys ttt gcc tca gca gta gaa tgt ctg cag cag atc agc aca aca ttt acc 4824 Phe Ala Ser Ala Val Glu Cys Leu Gln Gln Ile Ser Thr Thr Phe Thr cca tca gac aaa ctt aag gtc atc cag cag act ttt gag gag atc tct 4872 Pro Ser Asp Lys Leu Lys Val Ile Gln Gln Thr Phe Glu Glu Ile Ser SUBSTITUTE SHEET (RULE 26) cag agt gtc ctg gcg tca ctc cac gaa gac ttc ttg tgg tcc atg gat 4920 Gln Ser Val Leu Ala Ser Leu His Glu Asp Phe Leu Trp Ser Met Asp gac ttg ttt cct gtt ttc tta tat gtg gtg cta cgg gcc agg att agg 4968 Asp Leu Phe Pro Val Phe Leu Tyr Val Val Leu Arg Ala Arg Ile Arg aat tta ggc tct gag gta cac ctc att gag gat cta atg gac ccc tat 5016 Asn Leu Gly Ser Glu Val His Leu Ile Glu Asp Leu Met Asp Pro Tyr ctt cag cat ggg gaa cag ggt ata atg ttc acc acc ttg aag gca tgt 5064 Leu Gln His Gly Glu Gln Gly Ile Met Phe Thr Thr Leu Lys Ala Cys tac tac cag att cag cgt gag aag ctt aac tag gctgcataac agcttgaaaa 5117 Tyr Tyr Gln Ile Gln Arg Glu Lys Leu Asn ctggattatc tactacagag tgttataaca ccatctggag tcttcctgta gtggcaaaaa 5177 agaacagtgt tgaaattgga aaggactttg tgttatttag gttgttagaa tgagccttac 5237 caataataag agccctgagc ccagaaaaaa ggactgtata gtttaaaggg aggattgaaa 5297 gggaggtaaa aaatcagatt agaccagttc ttggcctatg ataagttcca aaaataccat 5357 ttatctacta tttgaaaaaa gaagaggata tcccttccta cagtaaaggg tatgtcagct 5417 acatgaagtt gtaagaaaag cttccagtag agcttcttat attaaagaag ttgatggata 5477 tttttgaatt tctggtttgc ctgaatccac ctgcagttac cccgatccgt ttgcaagaac 5537 cagatcgtac ttgaaactat agtggccaca ctctgccttc ctgagtccct tccagtcatg 5597 tgtgcatcat gtctctttgc caagggaggg gagaaaggaa cttttaaact gcagttttaa 5657 ctttttctaa gctgtttctt gatgggagag gttctgtgca aaactaccac attctgtccc 5717 caaaatgtgg aatgcatcca aataggagtc ttctgcctct taacttaaaa gaacatagga 5777 attttgtttt tggtttcttt atcatgctac agagagtgaa tacactggaa ttcagacacc 5837 gactctgagc tgctaggaac ctcatttgtc catgtgcaaa cgctgtattc caaggcctgt 5897 gaatggcagc ctgaggaagt tttgcatgca ggctgtgttt tcgagcagga ctaacaactg 5957 ggaaataagc aaaaaactgc atcgatcccc agcctggtgt tgttcttccc tatacttcac 6017 actgaactca ggatgggaag aaaaaggaaa caagctttgg ctttttccat ctcaaaagta 6077 ttgtggcacc tcaacatttc agtgttttgc tttttaaaaa atgccctatt gtaagttgtt 6137 ggtttatact gtataagtaa cactagtagc tgttttgaat aacataggtg ctcttcctca 6197 tctcatctcc tacaccgtgg tgagcataca gagtgtcctg atttgtgtta agtgactgag 6257 aagatgttaa ttacttttga aaaaggatca tggtttttgc tctactttat aatcaagaca 6317 agtgtttatt aaaatactgt tttggaatgt tggctgtaat gtaacagcaa ttttcataat 6377 aaaaggcatt catcttt 6394 <210> 2 <211> 1657 <212> PRT
<213> Homo Sapiens <400> 2 Met Asp Ser Lys Lys Arg Ser Ser Thr Glu Ala Glu Gly Ser Lys Glu Arg Gly Leu Val His Ile Trp Gln Ala Gly Ser Phe Pro Ile Thr Pro Glu Arg Leu Pro Gly Trp Gly Gly Lys Thr Val Leu Gln Ala Ala Leu Gly Val Lys His Gly Val Leu Leu Thr Glu Asp Gly Glu Val Tyr Ser Phe Gly Thr Leu Pro Trp Arg Ser Gly Pro Val Glu Ile Cys Pro Ser Ser Pro Ile Leu Glu Asn Ala Leu Val Gly Gln Tyr Val Ile Thr Val SUBSTITUTE SHEET (RULE 26) Ala Thr Gly Ser Phe His Ser Gly Ala Val Thr Asp Asn Gly Val Ala Tyr Met Trp Gly Glu Asn Ser Ala Gly Gln Cys Ala Val Ala Asn Gln Gln Tyr Val Pro Glu Pro Asn Pro Val Ser Ile Ala Asp Ser Glu Ala Ser Pro Leu Leu Ala Val Arg Ile Leu Gln Leu Ala Cys Gly Glu Glu His Thr Leu Ala Leu Ser Ile Ser Arg Glu Ile Trp Ala Trp Gly Thr Gly Cys Gln Leu Gly Leu Ile Thr Thr Ala Phe Pro Val Thr Lys Pro Gln Lys Val Glu His Leu Ala Gly Arg Val Val Leu Gln Val Ala Cys Gly Ala Phe His Ser Leu Ala Leu Val Gln Cys Leu Pro Ser Gln Asp Leu Lys Pro Val Pro Glu Arg Cys Asn Gln Cys Ser Gln Leu Leu Ile Thr Met Thr Asp Lys Glu Asp His Val Ile Ile Ser Asp Ser His Cys Cys Pro Leu Gly Val Thr Leu Thr Glu Ser Gln Ala Glu Asn His Ala Ser Thr Ala Leu Ser Pro Ser Thr Glu Thr Leu Asp Arg Gln Glu Glu Val Phe Glu Asn Thr Leu Val Ala Asn Asp Gln Ser Val Ala Thr Glu Leu Asn Ala Val Ser Ala Gln Ile Thr Ser Ser Asp Ala Met Ser Ser Gln Gln Asn Val Met Gly Thr Thr Glu Ile Ser Ser Ala Arg Asn Ile Pro Ser Tyr Pro Asp Thr Gln Ala Val Asn Glu Tyr Leu Arg Lys Leu Ser Asp His Ser Val Arg Glu Asp Ser Glu His Gly Glu Lys Pro Met Pro Ser Gln Pro Leu Leu Glu Glu Ala Ile Pro Asn Leu His Ser Pro Pro Thr Thr Ser Thr Ser Ala Leu Asn Ser Leu Val Val Ser Cys Ala Ser Ala Val Gly Val Arg Val Ala Ala Thr Tyr Glu Ala Gly Ala Leu Ser Leu Lys Lys Val Met Asn Phe Tyr Ser Thr Thr Pro Cys Glu Thr Gly Ala Gln Ala Gly Ser Ser Ala Ile Gly Pro Glu Gly Leu Lys Asp Ser Arg Glu Glu Gln Val Lys Gln Glu Ser Met Gln Gly Lys Lys Ser Ser Ser Leu Val Asp Ile Arg Glu Glu Glu Thr Glu Gly Gly Ser Arg Arg Leu Ser Leu Pro Gly Leu Leu Ser Gln Val Ser Pro Arg Leu Leu Arg Lys Ala Ala Arg Val Lys Thr Arg Thr Val Val Leu Thr Pro Thr Tyr Ser Gly Glu Ala Asp Ala Leu Leu Pro Ser Leu Arg Thr Glu Val Trp Thr Trp Gly Lys Gly Lys Glu Gly Gln Leu Gly His Gly Asp Val Leu Pro Arg Leu Gln Pro Leu Cys Val Lys Cys Leu Asp Gly Lys Glu Val Ile His Leu Glu Ala Gly Gly Tyr His Ser Leu Ala Leu Thr Ala Lys Ser Gln Val Tyr Ser Trp Gly Ser Asn Thr Phe Gly Gln Leu Gly SUBSTITUTE SHEET (RULE 26) His Ser Asp Phe Pro Thr Thr Val Pro Arg Leu Ala Lys Ile Ser Ser Glu Asn Gly Val Trp Ser Ile Ala Ala Gly Arg Asp Tyr Ser Leu Phe Leu Val Asp Thr Glu Asp Phe Gln Pro Gly Leu Tyr Tyr Ser Gly Arg Gln Asp Pro Thr Glu Gly Asp Asn Leu Pro Glu Asn His Ser Gly Ser Lys Thr Pro Val Leu Leu Ser Cys Ser Lys Leu Gly Tyr Ile Ser Arg Val Thr Ala Gly Lys Asp Ser Tyr Leu Ala Leu Val Asp Lys Asn Ile Met Gly Tyr Ile Ala Ser Leu His Glu Leu Ala Thr Thr Glu Arg Arg Phe Tyr Ser Lys Leu Ser Asp Ile Lys Ser Gln Ile Leu Arg Pro Leu Leu Ser Leu Glu Asn Leu Gly Thr Thr Thr Thr Val Gln Leu Leu Gln Glu Val Ala Ser Arg Phe Ser Lys Leu Cys Tyr Leu Ile Gly Gln His Gly Ala Ser Leu Ser Ser Phe Leu His Gly Val Lys Glu Ala Arg Ser Leu Val Ile Leu Lys His Ser Ser Leu Phe Leu Asp Ser Tyr Thr Glu Tyr Cys Thr Ser Ile Thr Asn Phe Leu Val Met Gly Gly Phe Gln Leu Leu Ala Lys Pro Ala Ile Asp Phe Leu Asn Lys Asn Gln Glu Leu Leu Gln Asp Leu Ser Glu Val Asn Asp Glu Asn Thr Gln Leu Met Glu Ile Leu Asn Thr Leu Phe Phe Leu Pro Ile Arg Arg Leu His Asn Tyr Ala Lys Val Leu Leu Lys Leu Ala Thr Cys Phe Glu Val Ala Ser Pro Glu Tyr Gln Lys Leu Gln Asp Ser Ser Ser Cys Tyr Glu Cys Leu Ala Leu His Leu Gly Arg Lys Arg Lys Glu Ala Glu Tyr Thr Leu Gly Phe Trp Lys Thr Phe Pro Gly Lys Met Thr Asp Ser Leu Arg Lys Pro Glu Arg Arg Leu Leu Cys Glu Ser Ser Asn Arg Ala Leu Ser Leu Gln His Ala Gly Arg Phe Ser Val Asn Trp Phe Ile Leu Phe Asn Asp Ala Leu Val His Ala Gln Phe Ser Thr His His Val Phe Pro Leu Ala Thr Leu Trp Ala Glu Pro Leu Ser Glu Glu Ala Gly Gly Val Asn Gly Leu Lys Ile Thr Thr Pro Glu Glu Gln Phe Thr Leu Ile Ser Ser Thr Pro Gln Glu Lys Thr Lys Trp Leu Arg Ala Ile Ser Gln Ala Val Asp Gln Ala Leu Arg Gly Met Ser Asp Leu Pro Pro Tyr Gly Ser Gly Ser Ser Val Gln Arg Gln Glu Pro Pro Ile Ser Arg Ser Ala Lys Tyr Thr Phe Tyr Lys Asp Pro Arg Leu Lys Asp Ala Thr Tyr Asp Gly Arg Trp Leu Ser Gly Lys Pro His Gly Arg Gly Val Leu Lys Trp Pro Asp Gly Lys Met Tyr Ser Gly Met Phe Arg Asn Gly Leu Glu Asp Gly Tyr Gly Glu Tyr Arg SUBSTITUTE SHEET (RULE 26) Ile Pro Asn Lys Ala Met Asn Lys Glu Asp His Tyr Val Gly His Trp Lys Glu Gly Lys Met Cys Gly Gln Gly Val Tyr Ser Tyr Ala Ser Gly Glu Val Phe Glu Gly Cys Phe Gln Asp Asn Met Arg His Gly His Gly Leu Leu Arg Ser Gly Lys Leu Thr Ser Ser Ser Pro Ser Met Phe Ile Gly Gln Trp Val Met Asp Lys Lys Ala Gly Tyr Gly Val Phe Asp Asp Ile Thr Arg Gly Glu Lys Tyr Met Gly Met Trp Gln Asp Asp Val Cys Gln Gly Asn Gly Val Val Val Thr Gln Phe Gly Leu Tyr Tyr Glu Gly Asn Phe His Leu Asn Lys Met Met Gly Asn Gly Val Leu Leu Ser Glu Asp Asp Thr Ile Tyr Glu Gly Glu Phe Ser Asp Asp Trp Thr Leu Ser Gly Lys Gly Thr Leu Thr Met Pro Asn Gly Asp Tyr Ile Glu Gly Tyr Phe Ser Gly Glu Trp Gly Ser Gly Ile Lys Ile Thr Gly Thr Tyr Phe Lys Pro Ser Leu Tyr Glu Ser Asp Lys Asp Arg Pro Lys Val Phe Arg Lys Leu Gly Asn Leu Ala Val Pro Ala Asp Glu Lys Trp Lys Ala Val Phe Asp Glu Cys Trp Arg Gln Leu Gly Cys Glu Gly Pro Gly Gln Gly Glu Val Trp Lys Ala Trp Asp Asn Ile Ala Val Ala Leu Thr Thr Ser Arg Arg Gln His Arg Asp Ser Pro Glu Ile Leu Ser Arg Ser Gln Thr Gln Thr Leu Glu Ser Leu Glu Phe Ile Pro Gln His Val Gly Ala Phe Ser Val Glu Lys Tyr Asp Asp Ile Arg Lys Tyr Leu Ile Lys Ala Cys Asp Thr Pro Leu His Pro Leu Gly Arg Leu Val Glu Thr Leu Val Ala Val Tyr Arg Met Thr Tyr Val Gly Val Gly Ala Asn Arg Arg Leu Leu Gln Glu Ala Val Lys Glu Ile Lys Ser Tyr Leu Lys Arg Ile Phe Gln Leu Val Arg Phe Leu Phe Pro Glu Leu Pro Glu Glu Gly Ser Thr Ile Pro Leu Ser Ala Pro Leu Pro Thr Glu Arg Lys Ser Phe Cys Thr Gly Lys Ser Asp Ser Arg Ser Glu Ser Pro Glu Pro Gly Tyr Val Val Thr Ser Ser Gly Leu Leu Leu Pro Val Leu Leu Pro Arg Leu Tyr Pro Pro Leu Phe Met Leu Tyr Ala Leu Asp Asn Asp Arg Glu Glu Asp Ile Tyr Trp Glu Cys Val Leu Arg Leu Asn Lys Gln Pro Asp Ile Ala Leu Leu Gly Phe Leu Gly Val Gln Arg Lys Phe Trp Pro Ala Thr Leu Ser Ile Leu Gly Glu Ser Lys Lys Val Leu Pro Thr Thr Lys Asp Ala Cys Phe Ala Ser Ala Val Glu Cys Leu Gln Gln Ile Ser Thr Thr Phe Thr Pro Ser Asp Lys Leu Lys Val Ile Gln Gln Thr Phe Glu Glu Ile Ser Gln SUBSTITUTE SHEET (RULE 26) Ser Val Leu Ala Ser Leu His Glu Asp Phe Leu Trp Ser Met Asp Asp Leu Phe Pro Val Phe Leu Tyr Val Val Leu Arg Ala Arg Ile Arg Asn Leu Gly Ser Glu Val His Leu Ile Glu Asp Leu Met Asp Pro Tyr Leu Gln His Gly Glu Gln Gly Ile Met Phe Thr Thr Leu Lys Ala Cys Tyr Tyr Gln Ile Gln Arg Glu Lys Leu Asn <210> 3 <211> 49 <212> PRT
<213> Homo Sapiens <400> 3 Met Asp Ser Lys Lys Arg Ser Ser Thr Glu Ala Glu Gly Ser Lys Glu Arg Gly Leu Val His Ile Trp Gln Ala Gly Ser Phe Pro Ile Thr Pro Glu Arg Leu Pro Gly Trp Gly Gly Lys Thr Val Leu Gln Ala Pro Ser Glu <210> 4 <211> 6349 <212> DNA
<213> Mus musculus <220>
<221> CDS
<222> (124)..(5079) <400> 4 ccacgcgtcc ggcggtgcag tcgggctcgc gccgggagaa gagcgcggag ctgcgggagc 60 gtcaggtctt gagagagctt ttgctaatgg gatggtttgg tgatggagta ctcctcctga 120 ccg atg gac tca aag aag aaa agc tca aca gag gca gaa gga tcc aaa 168 Met Asp Ser Lys Lys Lys Ser Ser Thr Glu Ala Glu Gly Ser Lys gaa aga ggc cta gtc cat gtc tgg cag gca gga tcc ttt tct cta aca 216 Glu Arg Gly Leu Val His Val Trp Gln Ala Gly Ser Phe Ser Leu Thr cca gag agg ttg cca ggc tgg ggt gga aag aca gtt ctt cag gca gcc 264 Pro Glu Arg Leu Pro Gly Trp Gly Gly Lys Thr Val Leu Gln Ala Ala ctt ggt gtg agg cat gga gtt ctt ctg act gaa gat ggt gag gtc tac 312 Leu Gly Val Arg His Gly Val Leu Leu Thr Glu Asp Gly Glu Val Tyr agc ttt ggg act ctt ccc tgg aaa agt gaa tca gca gaa att tgt cca 360 Ser Phe Gly Thr Leu Pro Trp Lys Ser Glu Ser Ala Glu Ile Cys Pro agc agc ccc ctt cta gaa agt gcc ctg gtt ggg cat cat gtt att act 408 Ser Ser Pro Leu Leu Glu Ser Ala Leu Val Gly His His Val Ile Thr SUBSTITUTE SHEET (RULE 26) gtg gca aca ggg agc ttc cac agt gga gca gtg aca gag agc ggg gtg 456 Val Ala Thr Gly Ser Phe His Ser Gly Ala Val Thr Glu Ser Gly Val gtg tac atg tgg gga gag aat get gcc ggg cag tgt gcg gta get aac 504 Val Tyr Met Trp Gly Glu Asn Ala Ala Gly Gln Cys Ala Val Ala Asn cag cag tat gtg ccg gag ccg agt cct gtc agc att tct gac tcg gag 552 Gln Gln Tyr Val Pro Glu Pro Ser Pro Val Ser Ile Ser Asp Ser Glu acc agc ccg tca tta gca gtt agg att ctg caa ttg gca tgt ggc gag 600 Thr Ser Pro Ser Leu Ala Val Arg Ile Leu Gln Leu Ala Cys Gly Glu gaa cac aca ctg gca ttg tca ctc agc aga gag atc tgg gca tgg ggc 648 Glu His Thr Leu Ala Leu Ser Leu Ser Arg Glu Ile Trp Ala Trp Gly acc ggc tgt cag ctg ggc ctc atc acc acc act ttc cca gtg aca aag 696 Thr Gly Cys Gln Leu Gly Leu Ile Thr Thr Thr Phe Pro Val Thr Lys cca cag aag gtg gaa cac ctt get gga cga gtg gtg ctc cag gtg gcc 744 Pro Gln Lys Val Glu His Leu Ala Gly Arg Val Val Leu Gln Val Ala tgc ggt gca ttc cac agc ctt gca ctt gtg cag tgc ctc cct cct cag 792 Cys Gly Ala Phe His Ser Leu Ala Leu Val Gln Cys Leu Pro Pro Gln gat ctg aag cca gtc cca gag aga tgc aat cag tgc agc cag ctg ctc 840 Asp Leu Lys Pro Val Pro Glu Arg Cys Asn Gln Cys Ser Gln Leu Leu atc acc atg aca gac aaa gag gac cat gtg ata ata tcg gac agc cat 888 Ile Thr Met Thr Asp Lys Glu Asp His Val Ile Ile Ser Asp Ser His tgc tgc cct tta ggt gtg aca ttg tcc gag tct caa gca gaa aag cat 936 Cys Cys Pro Leu Gly Val Thr Leu Ser Glu Ser Gln Ala Glu Lys His gcc agc cct get ccc agc cct cac cca gag gca ctg gat gag cag gga 984 Ala Ser Pro Ala Pro Ser Pro His Pro Glu Ala Leu Asp Glu Gln Gly gag gtg ttt gag aac acg gtg gta gaa get gaa ctg aac atg gga agc 1032 Glu Val Phe Glu Asn Thr Val Val Glu Ala Glu Leu Asn Met Gly Ser agt cag acc aca agt ggc agt gcc att tcc acc cag cag aac atc gtg 1080 Ser Gln Thr Thr Ser Gly Ser Ala Ile Ser Thr Gln Gln Asn Ile Val gga aca get gaa gtg tct tct gcc aga aca get ccg tca tac cca gac 1128 Gly Thr Ala Glu Val Ser Ser Ala Arg Thr Ala Pro Ser Tyr Pro Asp acc cat gcg gta act gca tac ctg cag aag ctg tca gag cat tcg atg 1176 Thr His Ala Val Thr Ala Tyr Leu Gln Lys Leu Ser Glu His Ser Met SUBSTITUTE SHEET (RULE 26) agg gag aac cat gag cct gga gaa aag cca ccc cag gtc cag cct ctt 1224 Arg Glu Asn His Glu Pro Gly Glu Lys Pro Pro Gln Val Gln Pro Leu gta gaa gaa gca gtt cct gat ctt cac agt cca cca acc aca agc acc 1272 Val Glu Glu Ala Val Pro Asp Leu His Ser Pro Pro Thr Thr Ser Thr tca gcc ctc aac agc ttg gtg gtc tcc tgt gca tct get gtt ggt gtc 1320 Ser Ala Leu Asn Ser Leu Val Val Ser Cys Ala Ser Ala Val Gly Val aga gtg get gcc acc tat gaa get ggg gcc ttg tct ctc aag aaa gtt 1368 Arg Val Ala Ala Thr Tyr Glu Ala Gly Ala Leu Ser Leu Lys Lys Val atg aac ttt tac agc act gcc ccc tgc gag acg gca get cag tcg ggc 1416 Met Asn Phe Tyr Ser Thr Ala Pro Cys Glu Thr Ala Ala Gln Ser Gly agt gcc tcc aca ggc cca gaa agt ctg aaa gat ctc cga gaa gag cag 1464 Ser Ala Ser Thr Gly Pro Glu Ser Leu Lys Asp Leu Arg Glu Glu Gln gtg aaa cag gag tca ctg caa ggg aag aaa agc tca agt ctc atg gac 1512 Val Lys Gln Glu Ser Leu Gln Gly Lys Lys Ser Ser Ser Leu Met Asp atc aga gag gaa gag tcg gag gga ggg agt cga aga ctc tcc ctc cca 1560 Ile Arg Glu Glu Glu Ser Glu Gly Gly Ser Arg Arg Leu Ser Leu Pro ggg ttg ttg tcg caa gtt tcc ccc agg ctc tta agg aag get gcg agg 1608 Gly Leu Leu Ser Gln Val Ser Pro Arg Leu Leu Arg Lys Ala Ala Arg gtg aaa act cgg aca gtg gtt ctg act ccc aca tac agt gga gaa gca 1656 Val Lys Thr Arg Thr Val Val Leu Thr Pro Thr Tyr Ser Gly Glu Ala gat gcc ctt ctg cct tcc ctg agg aca gag gtg tgg acc tgg ggg aaa 1704 Asp Ala Leu Leu Pro Ser Leu Arg Thr Glu Val Trp Thr Trp Gly Lys ggc aag gaa ggg cag cta ggg cac ggc gac gtc ctg ccc agg ctt cag 1752 Gly Lys Glu Gly Gln Leu Gly His Gly Asp Val Leu Pro Arg Leu Gln ccg ttg tgt gtc aag tgt ctg gat ggt aaa gag gta atc cac ctg gag 1800 Pro Leu Cys Val Lys Cys Leu Asp Gly Lys Glu Val Ile His Leu Glu gcg ggc ggc tcc cac tcc ctc gca ctc act gcg aaa tct cag gtt tac 1848 Ala Gly Gly Ser His Ser Leu Ala Leu Thr Ala Lys Ser Gln Val Tyr tca tgg ggc agt aat acc ttt ggt cag ctt ggg cat tct gag ttt cca 1896 Ser Trp Gly Ser Asn Thr Phe Gly Gln Leu Gly His Ser Glu Phe Pro SUBSTITUTE SHEET (RULE 26) aca acg gtt cct cga ctc tca aag gtt agc agt gaa aat gga gtc tgg 1944 Thr Thr Val Pro Arg Leu Ser Lys Val Ser Ser Glu Asn Gly Val Trp agt gta get gca ggc caa gat tat tcc ttg ttt tta gtg gac acg gaa 1992 Ser Val Ala Ala Gly Gln Asp Tyr Ser Leu Phe Leu Val Asp Thr Glu gac ttc cag cct ggg ttg tat tac agt ggc cga cag gac cgt gca gaa 2040 Asp Phe Gln Pro Gly Leu Tyr Tyr Ser Gly Arg Gln Asp Arg Ala Glu ggt gat acc ctg cca gag aat ccc agt ggt aca aag act cca gta ctt 2088 Gly Asp Thr Leu Pro Glu Asn Pro Ser Gly Thr Lys Thr Pro Val Leu ctc tcc tgt agt aag ctt gga tac ata agc aga gta aca gca gga aaa 2136 Leu Ser Cys Ser Lys Leu Gly Tyr Ile Ser Arg Val Thr Ala Gly Lys gat agc tat cta gcc ttg gtg gat aag aac atc atg gga tac atc gcc 2184 Asp Ser Tyr Leu Ala Leu Val Asp Lys Asn Ile Met Gly Tyr Ile Ala agt ctc cat gag ttg get tct aca gaa aga cgg ttt tac tca aaa ctg 2232 Ser Leu His Glu Leu Ala Ser Thr Glu Arg Arg Phe Tyr Ser Lys Leu agc gaa atc aaa tca cag ata ctt agg cct ctt ctc agt tta gaa aat 2280 Ser Glu Ile Lys Ser Gln Ile Leu Arg Pro Leu Leu Ser Leu Glu Asn ttg ggc aca gtg acc act gtc cag ctg ttg cag gaa gtt gcc agc cgg 2328 Leu Gly Thr Val Thr Thr Val Gln Leu Leu Gln Glu Val Ala Ser Arg ttc agc aag ttg tgt tac ctc att ggg cag cat gga gcc tca cta agc 2376 Phe Ser Lys Leu Cys Tyr Leu Ile Gly Gln His Gly Ala Ser Leu Ser agc tac cta cag ggt atg aag gaa gcc agc agc ctg gtc atc atg aag 2424 Ser Tyr Leu Gln Gly Met Lys Glu Ala Ser Ser Leu Val Ile Met Lys cac tca agt ctt ttc ctg gac agc tac aca gag tac tgc aca tca gtt 2472 His Ser Ser Leu Phe Leu Asp Ser Tyr Thr Glu Tyr Cys Thr Ser Val tca aat ttc ctg gtt atg gga gga ttc cag ctt ctt get aag cct gcc 2520 Ser Asn Phe Leu Val Met Gly Gly Phe Gln Leu Leu Ala Lys Pro Ala att gat ttc cta aat aaa aac caa gaa ctc ttg caa gat ttg tca gaa 2568 Ile Asp Phe Leu Asn Lys Asn Gln Glu Leu Leu Gln Asp Leu Ser Glu gtg aat gat gag aac act cag ttg atg gaa atc ctg aac atg ctg ttt 2616 Val Asn Asp Glu Asn Thr Gln Leu Met Glu Ile Leu Asn Met Leu Phe ttc ttg cca atc aga cga ctt cat aat tat gca aaa gtt ttg cta aag 2664 Phe Leu Pro Ile Arg Arg Leu His Asn Tyr Ala Lys Val Leu Leu Lys SUBSTITUTE SHEET (RULE 26) ctt gcc act tgc ttt gaa gtg aca tct cca gag tat caa aag ctg cag 2712 Leu Ala Thr Cys Phe Glu Val Thr Ser Pro Glu Tyr Gln Lys Leu Gln gat tcc agt tct tgc tat gag tct ctt get ctc cat ctt ggc aag aag 2760 Asp Ser Ser Ser Cys Tyr Glu Ser Leu Ala Leu His Leu Gly Lys Lys agg aag gaa gca gag tac aca ctg agc ttc tgg aag acc ttt cct ggg 2808 Arg Lys Glu Ala Glu Tyr Thr Leu Ser Phe Trp Lys Thr Phe Pro Gly aaa atg acg gat tcc ttg agg aag cca gag cgc cgg ctg ctg tgt gag 2856 Lys Met Thr Asp Ser Leu Arg Lys Pro Glu Arg Arg Leu Leu Cys Glu agc agt aac cga gcc ctc tcc ctg cag cat gcc ggc agg ttt tct gtg 2904 Ser Ser Asn Arg Ala Leu Ser Leu Gln His Ala Gly Arg Phe Ser Val aat tgg ttc att ctc ttc aat gat gcc ctg gtc cat get cag ttc tct 2952 Asn Trp Phe Ile Leu Phe Asn Asp Ala Leu Val His Ala Gln Phe Ser aca cac cac gtg ttc cct ttg gcc aca ctc tgg gca gag cca cta tct 3000 Thr His His Val Phe Pro Leu Ala Thr Leu Trp Ala Glu Pro Leu Ser gaa gaa get ggt agc gtg aat ggc tta aag ata act aca cct gaa gaa 3048 Glu Glu Ala Gly Ser Val Asn Gly Leu Lys Ile Thr Thr Pro Glu Glu caa ttc aca ctc att tct tca aca ccc cag gaa aag acc aag tgg ctt 3096 Gln Phe Thr Leu Ile Ser Ser Thr Pro Gln Glu Lys Thr Lys Trp Leu cgg get att agc caa get gtg gat cag get ttg agg ggg acg tcc gat 3144 Arg Ala Ile Ser Gln Ala Val Asp Gln Ala Leu Arg Gly Thr Ser Asp ttc cca ctt tac gga ggc ggc agc agt gtt cag aga cag gaa cca ccc 3192 Phe Pro Leu Tyr Gly Gly Gly Ser Ser Val Gln Arg Gln Glu Pro Pro atc tca aga agt gcc aaa tac act ttc tac aag gat act cgc cta aag 3240 Ile Ser Arg Ser Ala Lys Tyr Thr Phe Tyr Lys Asp Thr Arg Leu Lys gat gcc act tac gat ggg cgc tgg ctt tca ggg aag cct cat ggc agg 3288 Asp Ala Thr Tyr Asp Gly Arg Trp Leu Ser Gly Lys Pro His Gly Arg ggt gtt ctg aag tgg cct gat gga aag atg tac tct ggc atg ttc agg 3336 Gly Val Leu Lys Trp Pro Asp Gly Lys Met Tyr Ser Gly Met Phe Arg aat ggc ttg gaa gat gga tat ggt gaa tac aga atc cct aac aag gcc 3384 Asn Gly Leu Glu Asp Gly Tyr Gly Glu Tyr Arg Ile Pro Asn Lys Ala SUBSTITUTE SHEET (RULE 26) ctg aac aaa gaa gac cat tat gta ggc cat tgg aaa gag ggg aaa atg 3432 Leu Asn Lys Glu Asp His Tyr Val Gly His Trp Lys Glu Gly Lys Met tgt ggg caa gga gtc tac agc tat gcc tct ggt gaa gtg ttt gaa ggc 3480 Cys Gly Gln Gly Val Tyr Ser Tyr Ala Ser Gly Glu Val Phe Glu Gly tgc ttt caa gat aac atg cgc cat ggg cat ggt ctg ctc cgg agt gga 3528 Cys Phe Gln Asp Asn Met Arg His Gly His Gly Leu Leu Arg Ser Gly aaa ctg act tct tct tct cct agc atg ttc att ggc cag tgg gta atg 3576 Lys Leu Thr Ser Ser Ser Pro Ser Met Phe Ile Gly Gln Trp Val Met gat aag aaa gca gga tat ggc gtc ttt gat gat atc acc agg gga gaa 3624 Asp Lys Lys Ala Gly Tyr Gly Val Phe Asp Asp Ile Thr Arg Gly Glu aag tac atg gga atg tgg cag gat gat gtg tgc caa ggg aat ggg gta 3672 Lys Tyr Met Gly Met Trp Gln Asp Asp Val Cys Gln Gly Asn Gly Val gta gtc acc cag ttt ggg tta tac tac gaa ggc aac ttc cac ctg aat 3720 Val Val Thr Gln Phe Gly Leu Tyr Tyr Glu Gly Asn Phe His Leu Asn aag atg atg gga aat ggg gtt ttg ctt tct gaa gat gat acc atc tat 3768 Lys Met Met Gly Asn Gly Val Leu Leu Ser Glu Asp Asp Thr Ile Tyr gaa gga gaa ttt tcc gat gac tgg aca ctt agt gga aag gga acg ctg 3816 Glu Gly Glu Phe Ser Asp Asp Trp Thr Leu Ser Gly Lys Gly Thr Leu act atg cca cat gga gat tat att gaa ggt tat ttt agt gga gaa tgg 3864 Thr Met Pro His Gly Asp Tyr Ile Glu Gly Tyr Phe Ser Gly Glu Trp gga tct ggg ata aaa atc act ggg acc tac ttc aaa cct agc ctg tat 3912 Gly Ser Gly Ile Lys Ile Thr Gly Thr Tyr Phe Lys Pro Ser Leu Tyr gag agc gat aag gac aag ccc aaa gcc ttc agg aag ctg ggg aac ctg 3960 Glu Ser Asp Lys Asp Lys Pro Lys Ala Phe Arg Lys Leu Gly Asn Leu gcc gtg gca gca gac gag aaa tgg aga gca gtg ttt gaa gaa tgc tgg 4008 Ala Val Ala Ala Asp Glu Lys Trp Arg Ala Val Phe Glu Glu Cys Trp cac cag ctg ggc tgt gag agc cca ggc caa ggg gag gtt tgg aaa gca 4056 His Gln Leu Gly Cys Glu Ser Pro Gly Gln Gly Glu Val Trp Lys Ala tgg gat aat att get gtg gcc ttg acc acg aac cgt cgc cag cat aaa 4104 Trp Asp Asn Ile Ala Val Ala Leu Thr Thr Asn Arg Arg Gln His Lys gac agt cca gaa ata cta agc cgc tct cag act cag acc ctg gag agt 4152 Asp Ser Pro Glu Ile Leu Ser Arg Ser Gln Thr Gln Thr Leu Glu Ser SUBSTITUTE SHEET (RULE 26) ct ttg gag tac att ccc cag cac att ggc gcc ttc tct gtg gag aaa tat 4200 Leu Glu Tyr Ile Pro Gln His Ile Gly Ala Phe Ser Val Glu Lys Tyr gat gac atc aag aag tat tta ata aag gcc tgt gat act cct ctg cac 4248 Asp Asp Ile Lys Lys Tyr Leu Ile Lys Ala Cys Asp Thr Pro Leu His cca ctg ggc agg ctt gtg gag acc ctg gtt gcg gtg tat aga atg aca 4296 Pro Leu Gly Arg Leu Val Glu Thr Leu Val Ala Val Tyr Arg Met Thr tat gtg ggt gta ggg gcc aac cgc cgg tta ctg cag gaa get gtg aag 4344 Tyr Val Gly Val Gly Ala Asn Arg Arg Leu Leu Gln Glu Ala Val Lys gag att aaa tct tat ctc aag agg att ttc cag ctt gtg agg ttc ttg 4392 Glu Ile Lys Ser Tyr Leu Lys Arg Ile Phe Gln Leu Val Arg Phe Leu ttt cct gag ctt cct gag gag ggc agc aca att cct ctt tct get cct 4440 Phe Pro Glu Leu Pro Glu Glu Gly Ser Thr Ile Pro Leu Ser Ala Pro ctg ccc act gga agg aga tcc ttc tgt act ggg aaa ttg gat tcc aga 4488 Leu Pro Thr Gly Arg Arg Ser Phe Cys Thr Gly Lys Leu Asp Ser Arg tcc gag tca cca gaa cca ggt tat gta gta aca agt tct ggc tta ctg 4536 Ser Glu Ser Pro Glu Pro Gly Tyr Val Val Thr Ser Ser Gly Leu Leu ctt ccg gtg ctg ctg ccg cgg ctc tac cca cct ctc ttc atg ctc tat 4584 Leu Pro Val Leu Leu Pro Arg Leu Tyr Pro Pro Leu Phe Met Leu Tyr gcc ctg gat aat gac cga gag gaa gac att tac tgg gaa tgt gtg ctt 4632 Ala Leu Asp Asn Asp Arg Glu Glu Asp Ile Tyr Trp Glu Cys Val Leu cga cta aac aag cag cca gat att get ctc ttg ggc ttc ctt gga gta 4680 Arg Leu Asn Lys Gln Pro Asp Ile Ala Leu Leu Gly Phe Leu Gly Val cag aaa aaa ttc tgg cca gcc acc ttg tca atc ctt gga gag agt aaa 4728 Gln Lys Lys Phe Trp Pro Ala Thr Leu Ser Ile Leu Gly Glu Ser Lys aag gtg ttg tca acc aca aag gat get tgc ttt gca tct gca gta gaa 4776 Lys Val Leu Ser Thr Thr Lys Asp Ala Cys Phe Ala Ser Ala Val Glu tgc ctg cag cag atc agc aca aca ttt act cca tca gac aag ctt aaa 4824 Cys Leu Gln Gln Ile Ser Thr Thr Phe Thr Pro Ser Asp Lys Leu Lys gtg atc cag cag acc ttt gaa gag atc tcc cag agt gtc ctt gca tcg 4872 Val Ile Gln Gln Thr Phe Glu Glu Ile Ser Gln Ser Val Leu Ala Ser SUBSTITUTE SHEET (RULE 26) ctg cag gag gac ttc ctc tgg tcc atg gat gac ttg ttc ccc gtc ttc 4920 Leu Gln Glu Asp Phe Leu Trp Ser Met Asp Asp Leu Phe Pro Val Phe tta tac gtg gtg ctg cgg gcc agg att cgg aac ttg ggc tct gaa gtt 4968 Leu Tyr Val Val Leu Arg Ala Arg Ile Arg Asn Leu Gly Ser Glu Val cac ctc att gag gat ctg atg gac ccc ttt ctc cag cat ggg gaa caa 5016 His Leu Ile Glu Asp Leu Met Asp Pro Phe Leu Gln His Gly Glu Gln ggc atc atg ttc acc acc ttg aag gcc tgt tac ttc cag att cag cgg 5064 Gly Ile Met Phe Thr Thr Leu Lys Ala Cys Tyr Phe Gln Ile Gln Arg gag aag ctt aac tag ggcgcctgac agcttgagga ccggattatc tgctgcggag 5119 Glu Lys Leu Asn gctacagcta tggcacaggc accgactgga ggctgatggg gcaaagaaca gtgttgaata 5179 cagaatggac ttttgtgcta ttttggttgt aatttctgag ccttactaat aattagagcc 5239 cagcatggaa aacatactgt atcattcaaa tggagactgg aaaaggagat agggatagag 5299 tagagtcttt ggcctgtgct gagatccaca cacctactta gaaaaggaaa ctggttaccc 5359 tttcctgtag tgaaagctct cagctccatg cagttccagg aaacctttcc aggaaagctg 5419 cttagatgaa aagaagttga tgactgtgtt taagctcctg gtttgtctaa ttccatttgc 5479 agttacccaa taccctttgg caaggagcag gttttacttg aaactgaagc agccatccct 5539 tgccttccta gacctctcgc tcccaggcac aagtgcagca tgctactttg ctaggggtgg 5599 gggtggggga gaagaagttt taaactgtag ttttaacctt ttgtaagccc ctttaccaag 5659 gcatttgtgg tcagagagct cccacggggt gactatgaca tcctggtccc ctcgtggaat 5719 gcatccacat aggatcttct gcctgctgac tgaaaagaac ataggaatac actggagtgc 5779 aaacactgcc gtgccaagct gctccaaacc tcactgatcc gaggcccact gcctacccag 5839 gaggcccact gcctacccag gaggcccgta agcttcttag cacaagcttt gtgtggagac 5899 tgaagatctg cacatgtgag gaagcaggga gctacagtgg ccctcagccc agtctgcggg 5959 tcttccctct acctcacact gaactcagaa gggaaggaag gagagacgca catgggattc 6019 tcccacctca gaagtattgt gacagcaccg cataaccacg gtttgctctt ttacaagcag 6079 cctcacaagt gtgggttgtg ggtgtgcgct ggagcagtgc cactcgtagc tgtttggata 6139 ccacaggtgc tcttccgtct catctgctgt actcggaggc gagcgcagtg gcctgactca 6199 tgggaaatga ctcagcaggc ggcaactact tttgaaaagg atcatgattt ccgagctact 6259 ttataatcaa gacaagcatt tgttaacata ctgttttgga atgttggctg taatgtaaca 6319 gcagttttca taataaatga cattcatctc 6349 <210> 5 <211> 1651 <212> PRT
<213> Mus musculus <400> 5 Met Asp Ser Lys Lys Lys Ser Ser Thr Glu Ala Glu Gly Ser Lys Glu Arg Gly Leu Val His Val Trp Gln Ala Gly Ser Phe Ser Leu Thr Pro Glu Arg Leu Pro Gly Trp Gly Gly Lys Thr Val Leu Gln Ala Ala Leu Gly Val Arg His Gly Val Leu Leu Thr Glu Asp Gly Glu Val Tyr Ser Phe Gly Thr Leu Pro Trp Lys Ser Glu Ser Ala Glu Ile Cys Pro Ser Ser Pro Leu Leu Glu Ser Ala Leu Val Gly His His Val Ile Thr Val Ala Thr Gly Ser Phe His Ser Gly Ala Val Thr Glu Ser Gly Val Val SUBSTITUTE SHEET (RULE 26) Tyr Met Trp Gly Glu Asn Ala Ala Gly Gln Cys Ala Val Ala Asn Gln Gln Tyr Val Pro Glu Pro Ser Pro Val Ser Ile Ser Asp Ser Glu Thr Ser Pro Ser Leu Ala Val Arg Ile Leu Gln Leu Ala Cys Gly Glu Glu His Thr Leu Ala Leu Ser Leu Ser Arg Glu Ile Trp Ala Trp Gly Thr Gly Cys Gln Leu Gly Leu Ile Thr Thr Thr Phe Pro Val Thr Lys Pro Gln Lys Val Glu His Leu Ala Gly Arg Val Val Leu Gln Val Ala Cys Gly Ala Phe His Ser Leu Ala Leu Val Gln Cys Leu Pro Pro Gln Asp Leu Lys Pro Val Pro Glu Arg Cys Asn Gln Cys Ser Gln Leu Leu Ile Thr Met Thr Asp Lys Glu Asp His Val Ile Ile Ser Asp Ser His Cys Cys Pro Leu Gly Val Thr Leu Ser Glu Ser Gln Ala Glu Lys His Ala Ser Pro Ala Pro Ser Pro His Pro Glu Ala Leu Asp Glu Gln Gly Glu Val Phe Glu Asn Thr Val Val Glu Ala Glu Leu Asn Met Gly Ser Ser Gln Thr Thr Ser Gly Ser Ala Ile Ser Thr Gln Gln Asn Ile Val Gly Thr Ala Glu Val Ser Ser Ala Arg Thr Ala Pro Ser Tyr Pro Asp Thr His Ala Val Thr Ala Tyr Leu Gln Lys Leu Ser Glu His Ser Met Arg Glu Asn His Glu Pro Gly Glu Lys Pro Pro Gln Val Gln Pro Leu Val Glu Glu Ala Val Pro Asp Leu His Ser Pro Pro Thr Thr Ser Thr Ser Ala Leu Asn Ser Leu Val Val Ser Cys Ala Ser Ala Val Gly Val Arg Val Ala Ala Thr Tyr Glu Ala Gly Ala Leu Ser Leu Lys Lys Val Met Asn Phe Tyr Ser Thr Ala Pro Cys Glu Thr Ala Ala Gln Ser Gly Ser Ala Ser Thr Gly Pro Glu Ser Leu Lys Asp Leu Arg Glu Glu Gln Val Lys Gln Glu Ser Leu Gln Gly Lys Lys Ser Ser Ser Leu Met Asp Ile Arg Glu Glu Glu Ser Glu Gly Gly Ser Arg Arg Leu Ser Leu Pro Gly Leu Leu Ser Gln Val Ser Pro Arg Leu Leu Arg Lys Ala Ala Arg Val Lys Thr Arg Thr Val Val Leu Thr Pro Thr Tyr Ser Gly Glu Ala Asp Ala Leu Leu Pro Ser Leu Arg Thr Glu Val Trp Thr Trp Gly Lys Gly Lys Glu Gly Gln Leu Gly His Gly Asp Val Leu Pro Arg Leu Gln Pro Leu Cys Val Lys Cys Leu Asp Gly Lys Glu Val Ile His Leu Glu Ala Gly Gly Ser His Ser Leu Ala Leu Thr Ala Lys Ser Gln Val Tyr Ser Trp Gly Ser Asn Thr Phe Gly Gln Leu Gly His Ser Glu Phe Pro Thr Thr Val Pro Arg Leu Ser Lys Val Ser Ser Glu Asn Gly Val Trp Ser SUBSTITUTE SHEET (RULE 26) Val Ala Ala Gly Gln Asp Tyr Ser Leu Phe Leu Val Asp Thr Glu Asp Phe Gln Pro Gly Leu Tyr Tyr Ser Gly Arg Gln Asp Arg Ala Glu Gly Asp Thr Leu Pro Glu Asn Pro Ser Gly Thr Lys Thr Pro Val Leu Leu Ser Cys Ser Lys Leu Gly Tyr Ile Ser Arg Val Thr Ala Gly Lys Asp Ser Tyr Leu Ala Leu Val Asp Lys Asn Ile Met Gly Tyr Ile Ala Ser Leu His Glu Leu Ala Ser Thr Glu Arg Arg Phe Tyr Ser Lys Leu Ser Glu Ile Lys Ser Gln Ile Leu Arg Pro Leu Leu Ser Leu Glu Asn Leu Gly Thr Val Thr Thr Val Gln Leu Leu Gln Glu Val Ala Ser Arg Phe Ser Lys Leu Cys Tyr Leu Ile Gly Gln His Gly Ala Ser Leu Ser Ser Tyr Leu Gln Gly Met Lys Glu Ala Ser Ser Leu Val Ile Met Lys His Ser Ser Leu Phe Leu Asp Ser Tyr Thr Glu Tyr Cys Thr Ser Val Ser Asn Phe Leu Val Met Gly Gly Phe Gln Leu Leu Ala Lys Pro Ala Ile Asp Phe Leu Asn Lys Asn Gln Glu Leu Leu Gln Asp Leu Ser Glu Val Asn Asp Glu Asn Thr Gln Leu Met Glu Ile Leu Asn Met Leu Phe Phe Leu Pro Ile Arg Arg Leu His Asn Tyr Ala Lys Val Leu Leu Lys Leu Ala Thr Cys Phe Glu Val Thr Ser Pro Glu Tyr Gln Lys Leu Gln Asp Ser Ser Ser Cys Tyr Glu Ser Leu Ala Leu His Leu Gly Lys Lys Arg Lys Glu Ala Glu Tyr Thr Leu Ser Phe Trp Lys Thr Phe Pro Gly Lys Met Thr Asp Ser Leu Arg Lys Pro Glu Arg Arg Leu Leu Cys Glu Ser Ser Asn Arg Ala Leu Ser Leu Gln His Ala Gly Arg Phe Ser Val Asn Trp Phe Ile Leu Phe Asn Asp Ala Leu Val His Ala Gln Phe Ser Thr His His Val Phe Pro Leu Ala Thr Leu Trp Ala Glu Pro Leu Ser Glu Glu Ala Gly Ser Val Asn Gly Leu Lys Ile Thr Thr Pro Glu Glu Gln Phe Thr Leu Ile Ser Ser Thr Pro Gln Glu Lys Thr Lys Trp Leu Arg Ala Ile Ser Gln Ala Val Asp Gln Ala Leu Arg Gly Thr Ser Asp Phe Pro Leu Tyr Gly Gly Gly Ser Ser Val Gln Arg Gln Glu Pro Pro Ile Ser Arg Ser Ala Lys Tyr Thr Phe Tyr Lys Asp Thr Arg Leu Lys Asp Ala Thr Tyr Asp Gly Arg Trp Leu Ser Gly Lys Pro His Gly Arg Gly Val Leu Lys Trp Pro Asp Gly Lys Met Tyr Ser Gly Met Phe Arg Asn Gly Leu Glu Asp Gly Tyr Gly Glu Tyr Arg Ile Pro Asn Lys Ala Leu Asn Lys Glu Asp His Tyr Val Gly His Trp Lys Glu Gly Lys Met Cys SUBSTITUTE SHEET (RULE 26) Gly Gln Gly Val Tyr Ser Tyr Ala Ser Gly Glu Val Phe Glu Gly Cys Phe Gln Asp Asn Met Arg His Gly His Gly Leu Leu Arg Ser Gly Lys Leu Thr Ser Ser Ser Pro Ser Met Phe Ile Gly Gln Trp Val Met Asp Lys Lys Ala Gly Tyr Gly Val Phe Asp Asp Ile Thr Arg Gly Glu Lys Tyr Met Gly Met Trp Gln Asp Asp Val Cys Gln Gly Asn Gly Val Val Val Thr Gln Phe Gly Leu Tyr Tyr Glu Gly Asn Phe His Leu Asn Lys Met Met Gly Asn Gly Val Leu Leu Ser Glu Asp Asp Thr Ile Tyr Glu Gly Glu Phe Ser Asp Asp Trp Thr Leu Ser Gly Lys Gly Thr Leu Thr Met Pro His Gly Asp Tyr Ile Glu Gly Tyr Phe Ser Gly Glu Trp Gly Ser Gly Ile Lys Ile Thr Gly Thr Tyr Phe Lys Pro Ser Leu Tyr Glu Ser Asp Lys Asp Lys Pro Lys Ala Phe Arg Lys Leu Gly Asn Leu Ala Val Ala Ala Asp Glu Lys Trp Arg Ala Val Phe Glu Glu Cys Trp His Gln Leu Gly Cys Glu Ser Pro Gly Gln Gly Glu Val Trp Lys Ala Trp Asp Asn Ile Ala Val Ala Leu Thr Thr Asn Arg Arg Gln His Lys Asp Ser Pro Glu Ile Leu Ser Arg Ser Gln Thr Gln Thr Leu Glu Ser Leu Glu Tyr Ile Pro Gln His Ile Gly Ala Phe Ser Val Glu Lys Tyr Asp Asp Ile Lys Lys Tyr Leu Ile Lys Ala Cys Asp Thr Pro Leu His Pro Leu Gly Arg Leu Val Glu Thr Leu Val Ala Val Tyr Arg Met Thr Tyr Val Gly Val Gly Ala Asn Arg Arg Leu Leu Gln Glu Ala Val Lys Glu Ile Lys Ser Tyr Leu Lys Arg Ile Phe Gln Leu Val Arg Phe Leu Phe Pro Glu Leu Pro Glu Glu Gly Ser Thr Ile Pro Leu Ser Ala Pro Leu Pro Thr Gly Arg Arg Ser Phe Cys Thr Gly Lys Leu Asp Ser Arg Ser Glu Ser Pro Glu Pro Gly Tyr Val Val Thr Ser Ser Gly Leu Leu Leu Pro Val Leu Leu Pro Arg Leu Tyr Pro Pro Leu Phe Met Leu Tyr Ala Leu Asp Asn Asp Arg Glu Glu Asp Ile Tyr Trp Glu Cys Val Leu Arg Leu Asn Lys Gln Pro Asp Ile Ala Leu Leu Gly Phe Leu Gly Val Gln Lys Lys Phe Trp Pro Ala Thr Leu Ser Ile Leu Gly Glu Ser Lys Lys Val Leu Ser Thr Thr Lys Asp Ala Cys Phe Ala Ser Ala Val Glu Cys Leu Gln Gln Ile Ser Thr Thr Phe Thr Pro Ser Asp Lys Leu Lys Val Ile Gln Gln Thr Phe Glu Glu Ile Ser Gln Ser Val Leu Ala Ser Leu Gln Glu Asp Phe Leu Trp Ser Met Asp Asp Leu Phe Pro Val Phe Leu SUBSTETUTE SHEET (RULE 26) TyrValVal Leu Ala Arg Ile Arg Leu Gly Ser ValHis Arg Asn Glu LeuIleGlu Asp Met Asp Pro Phe Gln His Gly GlnGly Leu Leu Glu IleMetPhe Thr Leu Lys Ala Cys Phe Gln Ile ArgGlu Thr Tyr Gln LysLeuAsn <210> 6 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Artificial Sequence: Synthesized Oligonucleotide <400> 6 cctagtcatc catgtgctgg 20 <210> 7 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Artificial Sequence: Synthesized Oligonucleotide <400> 7 tcccatacct gaccttccac 20 <210> 8 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Artificial Sequence: Synthesized Oligonucleotide <400> 8 cttgatagac tttctgtaaa gaag 24 <210> 9 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Artificial Sequence: Synthesized Oligonucleotide <400> 9 ggctacttgg acaaatctcc actg 24 <210> 10 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide Sk)BSTfTUTE SHEET (RULE 26) <400> 10 ggagagactg tgctcccaag 20 <210> 11 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 11 agccctcctt agccaatagc 20 <210> 12 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 12 taagcttagt gggcaggctc 20 <210> 13 <211> 22 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 13 ttcccactta acaaccatca ac 22 <210> 14 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 14 ccaatttggt taaatctata gggg 24 <210> 15 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 15 gacaatgcca gagtgtgctc 20 SUBSTITUTE SHEET (RULE 26) <210> 16 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 16 ccagcccttt gttagcagtc 20 <210> 17 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 17 cttcttcctg cctgtcaagg 20 <210> 18 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 18 ttgtacaatg cctcccttcc 20 <210> 19 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 19 agcccaacat gacacctttc 20 <210> 20 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 20 gattgcttgt tgcataaggg 20 <210> 21 SUBSTITUTE SHEET (RULE 26) <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 21 atacagcatg cgatgtcagg 20 <210> 22 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 22 ctggactccc actccttcac 20 <210> 23 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 23 gctagaagag cccagatttc c 21 <210> 24 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 24 tgactttgtg tgcctgtgtg 20 <210> 25 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 25 ataccctgga aaatctgggg 20 <210> 26 <211> 20 <212> DNA

SUBSTITUTE SHEET (RULE 26) <213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 26 tttgcgcatt atctctggtc 20 <210> 27 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 27 gtacgtatga aattcccccg 20 <210> 28 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 28 ttccgtctta ctcctgcacc 20 <210> 29 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 29 gccttaggat ccaattcctg 20 <210> 30 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 30 caatgatgta ctgatgaacc agc 23 <210> 31 <211> 20 <212> DNA
<213> Artificial Sequence SUBSTITUTE SHEET (RULE 26) <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 31 cctgatggtt taatggtggg 20 <210> 32 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 32 gcacatggca acaggttaag 20 <210> 33 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 33 tccttggcag aataaccctg 20 <210> 34 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 34 cccctaccac tccctttacc 20 <210> 35 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 35 ccagtggcta atagtacctg tcc 23 <210> 36 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized SUBSTITUTE SHEET (RULE 26) oligonucleotide <400> 36 tggatgcatg attcatttcc 20 <210> 37 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 37 tccttggctt tccaaatgtc 20 <210> 38 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 38 ctatcctggg gtctctgctg 20 <210> 39 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 39 tgctatcgaa atggttgctg 20 <210> 40 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 40 agctacgacc agcaaattcc 20 <210> 41 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide SUBSTITUTE SHEET (RULE 26) <400> 41 ~

ataggggtcc acctttcagg 20 <210> 42 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 42 aaggggatat gggcagagtc 20 <210> 43 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 43 aaatgcttgc ttggttttgg 20 <210> 44 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 44 aaagggcatc ttcattgcac 20 <210> 45 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 45 cacaagaggc agaaagagcc 20 <210> 46 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 46 aatgcttgat gaattgttgc c 21 SUBSTITUTE SHEET (RULE 26) <210> 47 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 47 atgatcatcc tcaccccagg 20 <210> 48 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 48 ttgaagattt atgcctgggg 20 <210> 49 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 49 tgaggtcaca cggctatcag 20 <210> 50 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 50 gtgtagtggg gctgatgtcc 20 <210> 51 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 51 tggctatgca aacattcagg 20 <210> 52 SUBSTITUTE SHEET (RULE 26) <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 52 aatgcaaaat accacacatg g 21 <210> 53 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 53 tcattggctt aaactgtggg 20 <210> 54 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 54 caacctaggg ttgatgcctg 20 <210> 55 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 55 catcttcgga aagcaaaacc . 20 <210> 56 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 56 ctttggggat atgactgcgt 20 <210> 57 <211> 26 <212> DNA

SUBSTITUTE SHEET (RULE 26) <213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 57 gtaaaagaat ttattaggga gaaaaa 26 <210> 58 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 58 ttcctctaac cccacatttt attc 24 <210> 59 <211> 26 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 59 tgcttttaaa atattaacca gctttg 26 <210> 60 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 60 tcagtcttgg cagttttggt c 21 <210> 61 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 61 ctgctgtatg ttgagcaggt g 21 <210> 62 <211> 20 <212> DNA
<213> Artificial Sequence SUBSTITUTE SHEET (RULE 26) <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 62 tggatgctcc actttgactg 20 <210> 63 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 63 ttaagaaccc ccttgagtgc 20 <210> 64 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 64 ttcctggtcc caaaattgac 20 <210> 65 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 65 cagggtgaaa ctacccaagc 20 <210> 66 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 66 ttttatgctt ttcaaccccc 20 <210> 67 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized SUBSTITUTE SHEET (RULE 26) oligonucleotide <400> 67 acacactttc tcgctgggac 20 <210> 68 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 68 tgatctgagc acaaaggctg 20 <210> 69 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 69 taaacagcgg tgggtagagc 20 <210> 70 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 70 aatgctcctt ttctcccact c 21 <210> 71 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 71 tgccaaattt ccaataatgc 20 <210> 72 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide SUBSTITUTE SHEET (RULE 26) <400> 72 taatggggac aaggaagcc 19 <210> 73 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 73 gctgaggcaa aacaagcatc 20 <210> 74 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 74 ccaaagacct gcactctgac 20 <210> 75 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 75 ctggcttggc tctctcctac 20 <210> 76 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 76 aaaaagcacg atcaaatggc 20 <210> 77 <211> 20 <212> DNA

<213> Artificial Sequence <220>

<223> Description of ArtificialSequence: synthesized oligonucleotide <400> 77 ggaagagcgt actcctgctg 20 SUBSTITUTE SHEET (RULE 26) <210> 78 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 78 gcaggagtac gctcttccac 20 <210> 79 <211> 22 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 79 gaacaaaatg tgctctaaag gc 22 <210> 80 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 80 tctttttctc tctggggcag 20 <210> 81 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 81 tgccttctgt gttttaccct g 21 <210> 82 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 82 gaagggaaca gggaaaagtg 20 <210> 83 SUBSTITUTE SHEET (RULE 26) <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: synthesized oligonucleotide <400> 83 ttacctccct ttcaatcctc c 21 <210> 84 <211> 545 <212> PRT
<213> Homo sapiens <400> 84 Met Asp Ser Lys Lys Arg Ser Ser Thr Glu Ala Glu Gly Ser Lys Glu Arg Gly Leu Val His Ile Trp Gln Ala Gly Ser Phe Pro Ile Thr Pro Glu Arg Leu Pro Gly Trp Gly Gly Lys Thr Val Leu Gln Ala Ala Leu Gly Val Lys His Gly Val Leu Leu Thr Glu Asp Gly Glu Val Tyr Ser Phe Gly Thr Leu Pro Trp Arg Ser Gly Pro Val Glu Ile Cys Pro Ser Ser Pro Ile Leu Glu Asn Ala Leu Val Gly Gln Tyr Val Ile Thr Val Ala Thr Gly Ser Phe His Ser Gly Ala Val Thr Asp Asn Gly Val Ala Tyr Met Trp Gly Glu Asn Ser Ala Gly Gln Cys Ala Val Ala Asn Gln Gln Tyr Val Pro Glu Pro Asn Pro Val Ser Ile Ala Asp Ser Glu Ala Ser Pro Leu Leu Ala Val Arg Ile Leu Gln Leu Ala Cys Gly Glu Glu 145 . 150 155 160 His Thr Leu Ala Leu Ser Ile Ser Arg Glu Ile Trp Ala Trp Gly Thr Gly Cys Gln Leu Gly Leu Ile Thr Thr Ala Phe Pro Val Thr Lys Pro Gln Lys Val Glu His Leu Ala Gly Arg Val Val Leu Gln Val Ala Cys Gly Ala Phe His Ser Leu Ala Leu Val Gln Cys Leu Pro Ser Gln Asp Leu Lys Pro Val Pro Glu Arg Cys Asn Gln Cys Ser Gln Leu Leu Ile Thr Met Thr Asp Lys Glu Asp His Val Ile Ile Ser Asp Ser His Cys Cys Pro Leu Gly Val Thr Leu Thr Glu Ser Gln Ala Glu Asn His Ala Ser Thr Ala Leu Ser Pro Ser Thr Glu Thr Leu Asp Arg Gln Glu Glu Val Phe Glu Asn Thr Leu Val Ala Asn Asp Gln Ser Val Ala Thr Glu Leu Asn Ala Val Ser Ala Gln Ile Thr Ser Ser Asp Ala Met Ser Ser Gln Gln Asn Val Met Gly Thr Thr Glu Ile Ser Ser Ala Arg Asn Ile Pro Ser Tyr Pro Asp Thr Gln Ala Val Asn Glu Tyr Leu Arg Lys Leu SUBSTITUTE SHEET (RULE 26) Ser Asp His Ser Val Arg Glu Asp Ser Glu His Gly Glu Lys Pro Met Pro Ser Gln Pro Leu Leu Glu Glu Ala Ile Pro Asn Leu His Ser Pro Pro Thr Thr Ser Thr Ser Ala Leu Asn Ser Leu Val Val Ser Cys Ala Ser Ala Val Gly Val Arg Val Ala Ala Thr Tyr Glu Ala Gly Ala Leu Ser Leu Lys Lys Val Met Asn Phe Tyr Ser Thr Thr Pro Cys Glu Thr Gly Ala Gln Ala Gly Ser Ser Ala Ile Gly Pro Glu Gly Leu Lys Asp Ser Arg Glu Glu Gln Val Lys Gln Glu Ser Met Gln Gly Lys Lys Ser Ser Ser Leu Val Asp Ile Arg Glu Glu Glu Thr Gly Arg Gln Ser Lys Thr Leu Pro Pro Trp Ile Val Val Thr Ser Phe Pro Gln Ala Leu Lys Lys Gly Cys Thr Gly Glu Asn Glu Asp Ser Gly Ser Asp Pro His Ile Gln Trp Arg Ser Arg Cys Ala Pro Ala Phe Ser Glu Asn Arg Ser Val Asp Leu Gly Glu Arg Glu Gly Arg Ala Ala Gly Ala Arg Arg Cys Ser Ala SUBSTITUTE SHEET (RULE 26)

Claims (62)

WHAT IS CLAIMED IS:

1. An isolated nucleic acid that codes for a peptide having at least 75%
identity to all of an amino acid sequence selected from the group consisting of SEQ ID NO:2; SEQ
ID
NO:3; SEQ ID NO:5; SEQ ID NO:84; and, amino acids 372-1657 of SEQ ID NO:2.

2. The nucleic acid of claim 1 that codes for a peptide having about 80% or more sequence identity to the selected sequence.

3. The nucleic acid of claim 1 that codes for a peptide having about 85% or more sequence identity to the selected sequence.

4. The nucleic acid of claim 1 that codes for a peptide having about 90% or more sequence identity to the selected sequence.

5. The nucleic acid of claim 1 that codes a peptide having about 95% or more sequence identity to the selected sequence.

6. The nucleic acid of any one of claims 1-5, wherein the selected sequence is SEQ ID
NO:2.

7. The nucleic acid of claim 1, wherein the selected sequence is SEQ ID NO:3.

8. The nucleic acid of claim 1, wherein the selected sequence is SEQ ID NO:5.

9. The nucleic acid of claim 1, wherein the selected sequence is SEQ ID NO:84.

10. An isolated nucleic acid consisting essentially of a nucleotide sequence having at least 75% identity to all of a nucleotide sequence or a complementary sequence thereof, selected from the group consisting of SEQ ID NO:1; SEQ ID NO:4; nucleotides of SEQ ID NO:1; nucleotides 1225-5094 of SEQ ID NO:1; and, nucleotides 124-5076 of SEQ ID NO:4.

11. The nucleic acid of claim 10 having about 80% or more sequence identity to the selected sequence or complementary sequence thereof.

12. The nucleic acid of claim 10 having about 85% or more sequence identity to the selected sequence or complementary sequence thereof.

13. The nucleic acid of claim 10 having about 90% or more sequence identity to the selected sequence or complementary sequence thereof.

14. The nucleic acid of claim 10 having about 95% or more sequence identity to the selected sequence or complementary sequence thereof.

15. The nucleic acid of any one of claims 10-14, wherein the selected sequence is SEQ
ID NO:1.

16. The nucleic acid of any one of claims 10-14, wherein the selected sequence is SEQ
ID NO:4.

17. The nucleic acid of any one of claims 10-14, wherein the selected sequence is nucleotides 124-5094 of SEQ ID NO:1.

18. The nucleic acid of any one of claims 10-14, wherein the selected sequence is amino acids 124-5076 of SEQ ID NO:4.

19. The isolated nucleic acid of any one of claims 1-18 joined to a second nucleic acid, wherein the second nucleic acid is not naturally associated with the isolated nucleic acid.

20. A recombinant vector comprising a nucleic acid according to any one of claims 1-19.

21. A cell comprising a nucleic acid of claim 19 or a vector of claim 20.

22. An oligonucleotide of 6 to 75 nucleotides, wherein the oligonucleotide hybridizes to a nucleic acid according to any one of claims 1-18 or a complementary sequence thereof, under stringent conditions.

23. The oligonucleotide of claim 22 of about 10 to about 40 nucleotides.

24. The oligonucleotide of claim 22 of about 15 to about 30 nucleotides.

25. The oligonucleotide of claim 22 of about 15 to about 25 nucleotides.

26. The oligonucleotide of any one of claims 22-25 capable of hybridizing under stringent conditions to a nucleic acid encoding a peptide consisting of the sequence of SEQ
ID NO:3 or the complementary nucleic acid sequence thereof, but not to a nucleic acid encoding a peptide consisting of the sequence of SEQ ID NO:2 or the complementary nucleic acid sequence thereof.

27. The oligonucleotide of any one of claims 22-25 capable of hybridizing under stringent conditions to a nucleic acid encoding a peptide consisting of the sequence of SEQ
ID NO:84 or the complementary nucleic acid sequence thereof, but not to a nucleic acid encoding a peptide consisting of the sequence of SEQ ID NO:2 or the complementary nucleic acid sequence thereof.

28. The oligonucleotide of any one of claims 22-27 joined to a label.

29. A kit comprising two or more different oligonucleotides according to any one of claims 22-27 for use in nucleic acid amplification.

30. An isolated peptide comprising a sequence of amino acids coded by a nucleic acid according to any one of claims 1-19 or a recombinant vector according to claim 20.

31. A peptide consisting essentially of a sequence of at least 5 contiguous amino acids from a sequence selected from the group consisting of amino acids 1-46 of SEQ
ID NO:2;
amino acids 47-1657 of SEQ ID NO:2; SEQ ID NO:3; amino acids 43-49 of SEQ ID
NO:3;
SEQ ID NO:84; and amino acids 476 to 545 of SEQ ID NO:84.

32. A peptide comprising at least 5 contiguous amino acids from amino acids 43-49 of SEQ ID NO:3 or amino acids 476 to 545 of SEQ ID NO:84.

33. An antibody which binds a peptide according to any one of claims 30-32.

34. The antibody of claim 33 prepared by using a peptide according to any one of claims 30-32 as an antigen.

35. A non-human mammal comprising a mutated gene, wherein the gene but for the mutation would encode a protein having at least 75% sequence identity to all of SEQ ID
NO:2 or SEQ ID NO:5.

36. The mammal of claim 35, wherein the protein has at least 85% sequence identity to all of SEQ ID NO:1 or SEQ ID NO:2.

37. The mammal of claim 35 or 36, wherein the mutated gene does not express a protein having biological activity.

38. The mammal of claim 35, 36, or 37, wherein the mutated gene is incapable of expression of a protein.

39. The mammal of any one of claims 35-38, wherein the mammal is a rodent.

40. The mammal of claim 39, wherein the rodent is a mouse.

41. A method for the diagnosis of amyotrophic lateral sclerosis type 2 in a patient, comprising detecting the presence of a mutation in a gene that encodes a protein having at least 75% sequence identity to SEQ ID NO:2.

42. The method of claim 41, wherein the protein has at least about 90%
sequence identity to SEQ ID NO:2.

43. The method of claim 41, wherein the protein has at least about 95%
sequence identity to SEQ ID NO:2.

44. The method of claim 41, wherein the protein has at least about 97%
sequence identity to SEQ ID NO:2.

45. The method of claim 41, wherein the protein has essentially the sequence of SEQ ID
NO:2 but for the presence of the mutation.

46. The method of any one of claims 41-45, comprising detecting the presence of the mutation in a biological sample from the patient.

47. The method of any one of claims 41-46, wherein the detecting comprises comparing a sequence of the gene, a RNA transcript of the gene, and a cDNA made from the RNA
transcript, or a protein expressed by the gene from a human patient, to SEQ ID
NO:1, wherein a difference in sequence is indicative of mutation.

48. The method of claim 46, comprising contacting nucleic acids obtained from the biological sample or cDNA made from said nucleic acids, with one or more oligonucleotides according to any one of claims 22 to 28.

49. The method of claim 46, comprising detecting whether the one or more oligonucleotides hybridize to said nucleic acids or cDNA, under stringent conditions.

50. The method of claim 46, comprising amplification of nucleic acids or cDNA
to which two or more of said oligonucleotides hybridize, and determining the presence of an amplified product.

51. A method for the diagnosis of amyotrophic lateral sclerosis type 2, comprising detecting the presence or absence of a protein having at least 85% sequence identity to all of SEQ ID NO:2 in a patient.

52. A method for the diagnosis of amyotrophic lateral sclerosis type 2, comprising detecting the presence or absence of a protein having at least 95% sequence identity to all of SEQ ID NO:2 in a patient.

53. The method of claim 51, wherein the detecting comprises determining whether a protein having at least 85% sequence identity to all of SEQ ID NO:2 is present in a biological sample from the patient.

54. The method of claim 52, wherein the detecting comprises determining whether a protein having at least 95% sequence identity to all of SEQ ID NO:2 is present in a biological sample from the patient.

55. A method for the diagnosis of amyotrophic lateral sclerosis type 2, comprising detecting the presence or absence of a protein having at least 85% sequence identity to all of SEQ ID NO:3 or SEQ ID NO:84 in a biological sample from the patient.

56. A method for the diagnosis of amyotrophic lateral sclerosis type 2, comprising detecting the presence or absence of a protein having at least 95% sequence identity to all of SEQ ID NO:3 or SEQ ID NO:84 in a biological sample from the patient.

57. The method of any one of claims 51-56, comprising contacting an antibody according to any one of claims 33 or 34 with a biological sample from the patient and determining whether the antibody binds to protein in the sample.

58. A method of treatment of amyotrophic lateral sclerosis type 2, comprising administering a peptide, a nucleic acid, or a pharmaceutical composition comprising the peptide or nucleic acid to a patient in need thereof, wherein the peptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO:2 or a fragment thereof, and the nucleic acid codes for said peptide.

59. A method of treatment of amyotrophic lateral sclerosis type 2, comprising administering a peptide, a nucleic acid, or a pharmaceutical composition comprising the peptide or nucleic acid to a patient in need thereof, wherein the peptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO:2 or a fragment thereof, and the nucleic acid codes for said peptide.

60. A method of treatment of amyotrophic lateral sclerosis type 2, comprising administering a composition to a patient in need thereof, wherein the composition mimics the biological activity of the peptide of SEQ ID NO. 2.

61. The use of a peptide or a nucleic acid for preparation of a medicament for treatment of amyotrophic lateral sclerosis type 2, wherein the peptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO:2 or a fragment thereof, and the nucleic acid codes for said peptide.

62. The use of a peptide or a nucleic acid for preparation of a medicament for treatment of amyotrophic lateral sclerosis type 2, wherein the peptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO:2 or a fragment thereof, and the nucleic acid codes for said peptide.