CN1845994A - Ubiquitin-specific protease - Google Patents

Abstract

The present invention provides novel members of the family of deubiquitinating enzymes (DUBs) as well as biologically, diagnostically or therapeutically useful fragments, derivatives, and homologues thereof, which are useful in research, biological, clinical, therapeutic as well as prognostic and diagnostic purposes. Furthermore, the polynucleotides and polypeptides provided by the present invention are useful for the treatment and diagnosis of breast cancer, leukemia or brain disorders.

Description

The ubiquitin specific protease

Invention field

The present invention relates to ubiquitin specific protease (USP), relate in particular to the newcomer who takes off ubiquitin enzyme (DUB) family.

Background of invention

Ubiquitin be in all eukaryotic cells, find and its sequence at the protein of protozoon uptight 76 amino-acid residues to the vertebrates.This protein is kept in the degraded of the selectivity of the dependency ATP of various kinds of cell process such as cell protein, chromatin Structure, genetic expression adjusting, stress reaction and rrna is biological plays a significant role in taking place.With described little eukaryotic protein be can functionally modify or targeting proteins matter puting together of ubiquitin, be used for degrading by proteasome.Be similar to protein phosphorylation, the protein ubiquitination is dynamic, relates to enzyme (ubiquitin-conjugating enzyme) that adds ubiquitin and the enzyme of removing ubiquitin.The removal that ubiquitin is modified or take off the ubiquitin effect by ubiquitin specific protease (USP) by name or ubiquitin carboxyl terminal lytic enzyme (UCH) is carried out and be the important mechanisms of regulating this approach.These enzymes can cut the connection peptide bond as the ubiquitin of the part of precursor fusion rotein, discharge free ubiquitin part or cutting and put together ubiquitin and proteinic key (translation back).

Taking off the ubiquitin enzyme is the L-Cysteine HCL Anhydrous that identification and hydrolysis are positioned at the glycine peptide bond of ubiquitin carboxyl terminal.Exist two differences to take off ubiquitin enzyme (DUBs) family.First family is made up of the enzyme of about 25Kd and is representative with the UCHL1 among the mankind, UCHL3, Bap1 and UCH37 at present.This proteinoid belongs to the C12 family in the peptide enzyme classification.Second family is made up of bigger (800 to 2000 residues) protein, two similar zones are arranged between these protein, one of them zone comprises and may comprise two conservative histidine residues in related conservative halfcystine (halfcystine box) and another zone in catalyst mechanism, and one of them histidine residues also may relate to (Histidine box) in catalyst mechanism.These protein are at first described its feature and are belonged to the C19 family of MEROPS and be representative with USP in yeast.

Take off the ubiquitin enzyme and in cell, bring into play multiple effect, comprise that stablizing some substrate, the degraded of puting together with ubiquitin puts together the other substrate of (Ub) and the circulation in endocellular liberation monomer Ub storehouse through Ub.Some takes off the ubiquitin enzyme and removes Ub and therefore stoped the degraded (UBP2) of proteasome mediation property from the target protein of cell.Other take off, and the ubiquitin enzyme is removed Ub from the Ub-peptide degraded product that proteasome produced and therefore promoted the degraded (Doa-4) of proteasome mediation property

The effort that order-checking is explored to the different genes group has recently increased the sequence amount that demonstrates the conservative property feature of taking off the ubiquitin enzyme family.For example in yeast saccharomyces cerevisiae (S.cerevisiae), can identify a 17DUB enzyme family in the complete genome group that this primary yeast checked order.Several other protein that comprise conserved sequence motif (halfcystine box and Histidine box) in higher eucaryote source have also been identified.Therefore yet the great majority in these new sequences have been represented the member of brachymemma and these protein have been divided into really that a kind of to take off the ubiquitin family member be difficult.

Summary of the invention

The present invention in aspect first, provide the nucleotide sequence that comprises coding ubiquitin specific protease through DNA isolation, be selected from:

(a)SEQ?ID?No.2、SEQ?ID?No.6、SEQ.ID?No.10

(b) fragment of SEQ ID No.2, SEQ ID No.6, SEQ.ID No.10

(c) derivative of SEQ ID No.2, SEQ ID No.6, SEQ.ID No.10

(d) with SEQ ID No.2, the basic homologous sequence of SEQ ID No.6, SEQ.ID No.10.

In one aspect of the present invention, described DNA is selected from SEQ ID No.2, SEQ ID No.6 or SEQ.ID No.10.

In another aspect of the present invention, described fragment is the fragment of SEQ ID No.2, SEQ ID No.6 or SEQ.ID No.10 and has kept the special functionally active of ubiquitin.Described fragment can be long 50 continuous nucleotide at least, randomly for growing to few 75 or at least 100 s' continuous nucleotide.

Still in another aspect of the present invention, provide the derivative of SEQ ID No.2, SEQ ID No.6 or SEQ.ID No.10, wherein the disappearance of amino-acid residue, interpolation or the alternative aminoacid sequence that has produced the functional equivalent that keeps the special functionally active of ubiquitin in described aminoacid sequence.

Of the present invention aspect another in, provide with the basic homologous of nucleotide sequence that is selected from SEQ.ID No.2, SEQ ID No.6 or SEQ.ID No.10 and kept the nucleotide sequence of the special functionally active of ubiquitin.Homology percentage between basic homologous sequence and sequence SEQ.ID No.2, SEQ ID No.6 or the SEQ.ID No.10 wishes to be at least 80%, more wish to be at least 85%, be preferably at least 90%, more preferably be at least 95%, further more preferably be at least 99%.

Still in another aspect of the present invention, provide under the height stringent condition complementary nucleic acid sequence with arbitrary SEQ IDNo.2, SEQ ID No.6, SEQ.ID No.10 or its fragment, derivative or basic homologous sequence through DNA isolation hybridization.

In aspect second of the present invention, provide the separated polypeptide of ubiquitin specific protease, described polypeptide comprises and is selected from following aminoacid sequence:

(a)SEQ.ID.No.1、SEQ.ID.No.5、SEQ.ID?No.9

(b) fragment of SEQ.ID.No.1, SEQ.ID.No.5, SEQ.ID No.9

(c) derivative of SEQ.ID.No.1, SEQ.ID.No.5, SEQ.ID No.9

(d) with SEQ.ID.No.1, SEQ.ID.No.5, the basic homologous sequence of SEQ.ID No.9.

Preferred aspect of the present invention provides the separated polypeptide with aminoacid sequence shown in SEQ ID NO:1.Than non-patient with breast cancer's mammary tissue, huge amount ground exists described polypeptide or its fragment in patient with breast cancer's mammary tissue.According to this respect of the present invention, provide the novel polypeptide of human origin and this novel polypeptide fragment, derivative and the homologue useful biologically, in the diagnosis, in the treatment.

Another preferred aspect of the present invention provides the separated polypeptide with aminoacid sequence shown in SEQ ID NO:5.Than non-leukaemic's peripheral blood cells, described polypeptide or its fragment are in leukaemic's peripheral blood cells, and especially huge amount ground exists in the lymphoidocyte.According to this respect of the present invention, provide the novel polypeptide of human origin and this novel polypeptide fragment, derivative and the homologue useful biologically, in the diagnosis, in the treatment.

Another preferred aspect of the present invention provides the separated polypeptide with aminoacid sequence shown in SEQ ID NO:9.Than non-functional disorders of brain patient's cerebral tissue, at functional disorders of brain patient's cerebral tissue, especially huge amount ground exists described polypeptide or its fragment in amygdala, spinal cord and the olfactory bulb tissue.According to this respect of the present invention, provide the novel polypeptide of human origin and this novel polypeptide fragment, derivative and the homologue useful biologically, in the diagnosis, in the treatment.

The 3rd aspect of the present invention relates to and is used for the method diagnosed the mankind, this method need be measured and be selected from SEQ ID.No.1 from the mankind, the amount of the polypeptide of SEQ ID.No.5 or SEQ ID No.9, the fragment of described polypeptide, derivative or basic homologous sequence, if compare with polypeptide described in the healthy tissues or its segmental amount, the amount that described polypeptide or the existence of its fragment raise then suffers from certain disease to the mankind and has diagnostic significance.

One preferred aspect in, the method that is used for diagnosing human mammary cancer is included in the amount of people's mammary tissue measurement from fragment, derivative or the basic homologous sequence of the mankind's the polypeptide according to SEQ ID.No.1, described polypeptide, wherein the amount with polypeptide described in the healthy tissues compares, and the amount that described polypeptide existence raises then suffers from mammary cancer to the mankind and has diagnostic significance.

In aspect another is preferred, be used for the leukemic method of diagnosing human and be included in the human peripheral blood cell, especially measure the amount of fragment, derivative or basic homologous sequence in the lymphoidocyte from the mankind's polypeptide, described polypeptide according to SEQ ID.No.5, wherein the amount with normal circumference hemocyte especially polypeptide described in the lymphoidocyte compares, and the amount that described polypeptide existence raises then suffers from leukemia to the mankind and has diagnostic significance.

In aspect another is preferred, the method that is used for the diagnosing human functional disorders of brain is included in the amount that people's amygdala, spinal cord and olfactory bulb tissue are measured the fragment of the polypeptide according to SEQ ID.No.9 of human origin, described polypeptide, derivative or basic homologous sequence, wherein compare with polypeptide described in normal amygdala, spinal cord and the olfactory bulb tissue or its segmental amount, the amount that described polypeptide existence raises then suffers from functional disorders of brain to the mankind and has diagnostic significance.

Another aspect of the present invention provides and has been used to produce the fragment of aforesaid polypeptide, polypeptide fragment, derivative, homologue, variant and derivative and the method for their homologue.In the preferred embodiment of the present invention aspect this, the method that is used to produce aforementioned human polypeptide is provided, has been included under the condition that is enough to expression ubiquitin specific polypeptide in the host and cultivates the host cell of having integrated the expression vector that has comprised exogenous deutero-ubiquitin specificity polynucleotide; And reclaim expressed polypeptide.

According to another aspect of the present invention, especially provide product, composition and the method for utilizing aforesaid polypeptide and polynucleotide for research, biology, clinical and therapeutic purpose.

In some extra embodiment preferred, provide and the polypeptid specificity bonded antibody that comprises aminoacid sequence shown in SEQ ID NO:1, SEQ ID.No.5 or SEQ ID.No.9 or its fragment in this aspect of the invention.Thus, described antibody is selected people's ubiquitin specific polypeptide or its part height in some especially preferred embodiment.Further, fragment bonded antibody or its fragment are provided with aminoacid sequence shown in SEQ ID NO:1, SEQ ID.No.5 or SEQ ID.No.9.In a related aspect, provide the pharmaceutical composition that comprises this antibody.

In yet another aspect, the method for the treatment of experimenter's disease by antibody from significant quantity to the experimenter that use is provided, wherein said antibodies has polypeptide or its fragment or the part of aminoacid sequence shown in SEQ ID NO:1, SEQ ID.No.5 or the SEQID.No.9, wherein said disease be respectively by the existence of polypeptide SEQ ID NO:1, SEQ ID.No.5 or SEQ ID.No.9 in mammary tissue, peripheral blood cells or the cerebral tissue increase mediated or relevant with it.Also provide to be used for experimenter and the existence increase diseases associated of polypeptide or the diagnostic method of symptom, described method is included in the antibody that uses in the immune identification method in conjunction with the polypeptide with aminoacid sequence shown in SEQ ID NO:1, SEQ ID.No.5 or the SEQ ID.No.9 or its fragment or part.

Still in one aspect of the method, the invention provides can external breeding cell, be preferably mammalian cell, it more preferably is vertebrate cells, described cell can be grown in generation comprises the cultivation of polypeptide with aminoacid sequence shown in SEQ ID NO:1, SEQ ID.No.5 or the SEQ ID.No.9 or its fragment, derivative or basic homologous sequence, what wherein said cell randomly comprised the non-human transcriptional control sequence transcribes the control dna sequence dna, and the DNA that wherein this transcriptional control sequence control coding has the polypeptide of described aminoacid sequence transcribes.

In yet another aspect, the invention provides the method that is used to produce polypeptide, described method is included under the condition that is enough to this type of polypeptide of expression in host cell and cultivates such host cell, therefore caused the generation of expressed polypeptide and reclaimed expressed polypeptide that wherein said host cell has been integrated the expression vector that comprises external source deutero-ubiquitin specificity polynucleotide of the present invention in cell.

Still in another aspect of the present invention, authentication method and test kit are provided, be included in the above-mentioned normal expression or polypeptide or its segmental essential component that detect ubiquitin specificity polynucleotide of the present invention in the bodily tissue in patient source, described test kit for example comprise antibody or can with the oligonucleotide probe of multi-nucleotide hybrid of the present invention.In a preferred embodiment, this type of test kit also comprises the specification sheets that component in this type of test kit should be followed the program of use is described in detail in detail.

Another aspect relates to the nucleotide sequence that comprises SEQ ID NO:1, SEQ ID.No.5 or SEQ ID.No.9 or the pharmaceutical composition of its fragment, derivative or homologue.

In yet another aspect, the present invention relates to be used to identify the method for molecule, wherein said molecule can in conjunction with ubiquitin specific protease of the present invention and/or to regulate ubiquitin active or regulate can be in conjunction with the activity of the molecule of regulating the nucleotide sequence that ubiquitin transcribes or translate.These class methods are at for example U.S. Patent number 5,541,070; 5,567,317; 5,593,853; 5,670,326; 5,679,582; 5,856,083; 5,858,657; 5,866,341; 5,876,946; 5,989,814; 6,010,861; 6,020,141; Open in 6,030,779 and 6,043,024.All these patents are complete herein to be quoted as a reference.The molecule of identifying by these methods falls within the scope of the present invention equally.

Still in yet another aspect, the present invention relates to be used for to one or more tissues importing nucleic acid of the present invention of the experimenter of needs treatment and the protein that causes one or more described nucleic acid encodings by cell expressing in the described tissue and/or excretory method.

According to following description, others of the present invention, feature, advantage and aspect are conspicuous to those skilled in the art.Yet should understand following description and specific embodiment in explanation the preferred embodiments of the invention, only be used as explanation.Multiple change in disclosed spirit and scope of the invention and variation become apparent very soon by other parts of reading following description and reading content of the present invention for those skilled in the art.

The description of form

Table 1 (a): USP_N01: new splice variant-aminoacid sequence

Table 1 (a) has been described the SEQ ID.No.1 of the new splicing form of ubiquitin specific protease.The feature of this new splicing form is to be positioned at Pos1-Pos11, Pos267-Pos300,4 insertions of Pos361-Pos384 and Pos1243-Pos1437.

Table 1 (b): USP_N01: new splice variant-nucleotide sequence

Table 1 (b) has been described the SEQ ID.No.2 corresponding to the nucleotide sequence of table 1 (a).

Table 1 (c): DERWENT reference amino acid sequence AAU82706

Table 1 (c) is described the reference amino acid sequence SEQ ID.No.3 that has relatively crossed with the new splicing form of table 1 (a) as.

Table 1 (d): reference nucleotide sequence

Table 1 (d) has been described as the reference nucleotide sequence SEQ ID.No.4 corresponding to table 1 (c).

Table 2 (a): USP_N07: new splice variant-aminoacid sequence

Table 2 (a) has been described the SEQ ID.No.5 as the new splicing form of ubiquitin specific protease.The feature of this new splicing form is 1 insertion that is positioned at Pos14-Pos81.

Table 2 (b): USP_N07: new splice variant-nucleotide sequence

Table 2 (b) has been described as the nucleotide sequence SEQ ID.No.6 corresponding to table 2 (a).

Table 2 (c): DERWENT reference amino acid sequence AAU82714

Table 2 (c) has been described the reference amino acid sequence SEQ ID.No.7 that has relatively crossed with the new splicing form of table 2 (a) as.

Table 2 (d): reference nucleotide sequence

Table 2 (d) has been described the reference nucleotide sequence SEQ ID.No.8 corresponding to table 2 (c).

Table 3 (a): USP_N11: new splice variant-aminoacid sequence

Table 3 (a) has been described the SEQ ID.No.9 as the new splicing form of ubiquitin specific protease.The feature of this new splicing form is 1 insertion that is positioned at Pos12-Pos48.

Table 3 (b): USP_N11: new splice variant-nucleotide sequence

Table 3 (b) has been described as the nucleotide sequence SEQ ID.No.10 corresponding to table 3 (a).

Table 3 (c): DERWENT reference amino acid sequence AAU82713

Table 3 (c) has been described the reference amino acid sequence SEQ ID.No.11 that has relatively crossed with the new splicing form of table 3 (a) as.

Table 3 (d): reference nucleotide sequence

Table 3 (d) has been described as the reference nucleotide sequence SEQ ID.No.12 corresponding to table 3 (c).

Detailed Description Of The Invention

All patent applications, patent and the reference of quoting in the literary composition is complete here to be incorporated herein by reference.

When enforcement is of the present invention, used the multiple routine techniques in molecular biology, microbiology, the recombinant DNA.These technology are that " Current Protocols in Molecular Biology " I, II and III well-known and that edit at for example F.M.Ausubel in 1997 roll up; " Molecular Cloning:ALaboratory Manual " second edition of the Sambrook of press of cold spring harbor laboratory at cold spring port, New York in 1989 etc.; " DNA Cloning:A Practical Approach " I that D.N.Glover in 1985 edits and II volume; " the Oligonucleotide Synthesis " that M.L.Gait in 1984 edits; " the Nucleic AcidHybridization " of Hames in 1985 and Higgins; " the Transcription andTranslation " that Hames in 1984 and Higgins edit; " the Animal Cell Culture " that R.I.Freshney in 1986 edits; " the Immobilized Cells and Enzymes " of IRL press in 1986; " the A Practical Guide to Molecular Cloning " of Perbal in 1984; Academic Press, " Methods in Enzymology " series of books of Inc. press; Explain in detail in " the Gene Transfer Vectors for Mammalian Cells " that J.H.Miller of cold spring harbor laboratory in 1987 and M.P.Calos edit and " Methods in Enzymology " the 154th and 155 volumes of editing by Wu and Grossman and Wu respectively.

The present invention relates to identify the new splice variant that is called DUB or takes off the human ubiquitin specific protease of ubiquitin enzyme.New splice variant of the present invention provides in table 1 (a), 2 (a) and 3 (a), respectively corresponding aminoacid sequence SEQ ID.No.1, SEQ ID No.5 and SEQ ID.No.9.Corresponding nucleotide sequence SEQ ID No.2, SEQ ID No.6 and SEQ ID No.10 provide among 2 (b) and 3 (b) at table 1 (b).These sequences are respectively the splice variants of DERWENT reference sequences AAU82706, AAU82714 and AAU82713.

The present invention comprised with table 1,2 and 3 in the basic homologous nucleotide sequence of sequence and the aminoacid sequence that are provided.

Yet, be to be understood that prior to any aminoacid sequence disclosed in this invention be excluded.When using with regard to sequence herein, term " basic homologous " means certain sequence and have essentially identical 26S Proteasome Structure and Function when with its reference sequences comparison.When certain position in the reference sequences is identical amino acid or Nucleotide when occupying, molecule is in this position homology (promptly having identity in this position).Nucleotide sequence relatively in, homology still is present in such position, comprises in two molecules that the codeword triplet of Nucleotide comparing the identical amino acid because the degeneracy of genetic code has been encoded in described position.

Homology percentage between basic homologous sequence and the reference sequences wishes to be at least 80%, more wishes to be at least 85%, and preferably at least 90%, more preferably be at least 95%, further more preferably be at least 99%.

Sequence relatively uses Smith-Waterman sequence alignment algorithm to implement (to see for example nineteen ninety-five London Chapman ﹠amp; The Waterman of Hall press, " Introduction toComputational Biology:Maps, the sequences and genomes " ISBN0-412-99391-0 of M.S or Http:// www-to.usc.edu/software/seqaln/index.html).

Nucleotide sequence of the present invention also is included under the strict hybridization conditions sequence with nucleic acid array hybridizing of the present invention.Strict hybridization conditions is defined as at 7% sodium lauryl sulphate (SDS), 0.5MNaPO ₄, 50 ℃ of washings and in 2X SSC, 0.1%SDS, after 50 ℃ of washings, wish that more ground is at 7% sodium lauryl sulphate (SDS), 0.5M NaPO among the 1mM EDTA ₄, among the 1mM EDTA among 50 ℃ of washings and 1X SSC, the 0.1%SDS after 50 ℃ of washings, wish that more ground is at 7% sodium lauryl sulphate (SDS), 0.5M NaPO ₄, 50 ℃ of washings and in 0.5X SSC, 0.1%SDS after 50 ℃ of washings, among the 1mM EDTA preferably at 7% sodium lauryl sulphate (SDS), 0.5M NaPO ₄, 50 ℃ of washings and at 0.1X SSC among the 1mM EDTA are among the 0.1%SDS after 50 ℃ of washings, more preferably at 7% sodium lauryl sulphate (SDS), 0.5M NaPO ₄, among the 1mM EDTA 50 ℃ the washing and at 0.1X SSC, observed positive hybridization signal after 65 ℃ of washings among the 0.1%SDS.

Nucleotide sequence of the present invention can comprise open reading-frame (ORF) (Open Reading Frame, ORF) and comprise 5 ' untranslated zone (UTR) or its part.The present invention can also comprise the purposes that any promotor, enhanser, regulon, terminator and setting element and these elements are connected with heterologous gene.

The definition of the above homologous sequence that provides has comprised the fragment of the new splice variant of nucleic acid of the present invention or aminoacid sequence." fragment " mean have described new splice variant at least 5,10,15 or randomly have an any peptides molecule of at least 25,35 or 45 continuous amino acids.The fragment of nucleotide sequence comprises at least 50, randomly comprises at least 75 or at least 100 successive Nucleotide.

Yet the fragment that the present invention relates to will shall not be construed as and comprise prior to fragment disclosed in this invention.Fragment of the present invention can retaining protein one or more activity for example in conjunction with the ability of ubiquitin or hydrolysising peptide key, and the fragment that can produce the ubiquitin protease antibody as immunogen.Bioactive fragment can comprise structural domain or motif for example catalytic site, UBP or UCH mark, membrane-bound zone and be used for glycosylation, rely on protein kinase phosphorylation effect, protein kinase C phosphorylation, casein kinase i I phosphorylation, the Tyrosylprotein kinase phosphorylation of cAMP and cGMP, the site of effect of N-myristoylation and amidation.

Other possible fragment comprise catalytic site contain halfcystine or Histidine ubiquitin recognition site, ubiquitin binding site, for the important site of the interaction of subunit and to the important site of other function of carrying out this proteolytic enzyme at interior structural domain.This class formation territory or motif can identify that this class fragment can for example be extended to comprise 5,10,15,20,30,40,50 amino acid or to reach 100 amino acid by conventional computerized homology search utility on the both direction in functional site.

These fragments further can comprise the subfragment in above-mentioned ad hoc structure territory, and wherein these subfragments have kept the function of the structural domain that these subfragments originate.These zones can be identified by the well-known process that relates to the computerize homology analysis.The present invention also provides the fragment with immunogenicity feature.These fragments comprise carries the epi-position part in ubiquitin proteolytic enzyme and the variant.These peptides that carry epi-position are useful in conjunction with the regional or segmental antibody of the polypeptide of ubiquitin proteolytic enzyme for producing specificity.This class peptide can comprise at least 10,12, and at least 14, or about at least 15 between about 30 amino acid.The non-limiting example that can be used for producing the antigenic polypeptide of antibody includes but not limited to the peptide derived from the site, extracellular, and site, wherein said extracellular has comprised and has high antigenicity exponential zone (see Fig. 3, US 6,451,994).Yet the present invention also comprises the antibody (" intrabody ") that produces in the cell in peptide zone in the identification born of the same parents.These polypeptide that carry the ubiquitin proteolytic enzyme of epi-position can produce (Houghten, R.A. (1985) Proc.Natl.Acad.Sci.USA 82:5131-5135) by any conventional means.Synthesize at U.S. Patent number 4,631 in the time of a plurality of peptide, describe in 211.

Fragment can be independently (to merge with other amino acid or polypeptide) or can be in the bigger polypeptide.Several fragments further can comprise in a bigger polypeptide.In one embodiment, being designed for the fragment of expressing in the host can have and be blended in the additional areas that polypeptide zone and former polypeptide before the N-terminal heterology of ubiquitin proteolytic enzyme fragment are regional and be blended in this fragment carboxyl terminal.

Fragment can be used as the hybridization probe in cDNA library so that other gene that separates the gene of total length and have height sequence similarity and similar biologic activity.This class probe preferably has about at least 30 bases and can comprise for example about 30 to about 50 bases, and about 50 to about 100 bases, and about 100 to about 200 bases or above 200 bases.This probe can also be used to identify that cDNA clone or evaluation corresponding to total length transcript and genomic clone contain the Cloning of Entire Gene that comprises regulation domain and promoter region, exon and intron.The example of screening comprises by using the known dna sequence synthetic oligonucleotide probe to come the coding region of isolated genes.Have with gene order complementary sequence of the present invention through library that labeled oligonucleotide is used for screening people cDNA, genomic dna or mRNA to determine the member of library and this probe hybridization.

The present invention also comprises aminoacid sequence " derivative "." derivative " be with described aminoacid sequence in the relevant sequence of amino acid levels (as homologous sequence, wherein some naturally occurring amino acid is substituted by the synthetic amino acid surrogates) or in the relevant sequence (for example wherein molecule has and essentially identical shape of described aminoacid sequence and conformation) of space level.Therefore derivative comprises mutant, stand-in, mimotope thing (mimotopes), analogue, monomeric form and function equivalent.

Cause the reticent amino-acid residue disappearance that changes in the aminoacid sequence, add or substitute the difference expression gene product that has produced functional equivalent.Amino acid whose substitute can be based on the similarity of polarity, electric charge, solvability, hydrophobicity, wetting ability and/or the facultative feature of related residue.For example nonpolar (hydrophobicity) amino acid comprises L-Ala, leucine, Isoleucine, Xie Ansuan, proline(Pro), phenylalanine, tryptophane and methionine(Met); Polar neutral amino acid comprises glycine, Serine, Threonine, halfcystine, tyrosine, l-asparagine and glutamine; Positive charge (alkalescence) amino acid comprises arginine, Methionin and Histidine; And negative charge (acidity) amino acid comprises aspartic acid and L-glutamic acid.

Derivative can comprise such aminoacid sequence, and institute's alternate amino-acid residue is not the sort of by genetic code amino acids coding residue in its aminoacid sequence; In its aminoacid sequence, comprise surrogate; Wherein sophisticated peptide and another compound as the compound (as polyoxyethylene glycol) of polypeptide half life as described in improving or wherein merge extra amino acid such as leader sequence or secretion sequence or be used for the sequence of this mature polypeptide of purifying or preceding proteic sequence to mature polypeptide.

Known modification includes but not limited to acetylizing; acylation; the ADP ribosylation effect; amidation; flavine covalently bound; haemachrome molecule covalently bound; Nucleotide or nucleotide derivative covalently bound; lipid or lipid derivant covalently bound; phosphatidylinositols covalently bound; crosslinked; cyclic action; disulfide linkage forms; the demethylation effect; the formation of covalent cross-linking; the formation of Gelucystine; the formation of Pyrrolidonecarboxylic acid; formylation; the gamma-carboxylation effect; glycosylation; the formation of GPI anchor; hydroxylation; iodization; methylation; the myristoylation effect; oxygenizement; proteolysis processing, phosphorylation; the prenylation effect; racemization; the selenonyl effect; sulfation; the amino acid of transfer RNA (tRNA) mediation to proteinic interpolation for example arginyl turn into and ubiquitination.

This class is modified to that those skilled in the art know and describes in more detail in scientific literature.Several especially common modifications for example gamma-carboxylation effect, hydroxylation and the ADP ribosylation of glycosylation, fat connection, sulfation, glutaminic acid residue act in the teaching material on basis the most and describe, " the Proteins-Structure and Molecular Properties " the 2nd edition that edits as the New York W.H.Freeman and Company T.E.Creighton of press in 1993.Numerous " Posttranslational Covalent Modification of Proteins " the 1-12 pages or leaves that can for example edit about the more detailed summary of this theme at the nineteen eighty-three New York Academic Press B.C.Johnson of press; Seifter etc. obtain among (1992) Ann.N.Y.Acad.Sci.663:48-62 such as (1990) Meth.Enzymol.182:626-646 and Rattan.

Same well-known, polypeptide is complete wire always not, for example polypeptide can be the dendritic and polypeptide of branch because of ubiquitination and can be and possess branch or do not have branched ring-type, the result that this normally translates the post-treatment incident, comprise the natural process incident and natural do not take place by the incident due to people's generic operation.Cyclic, branched and branch annular polypeptide natural method that can be by untranslated and synthetic by synthetic method.

Modification can comprise that peptide main chain, amino acid side chain and aminoterminal or carboxyl terminal carry out in the optional position in polypeptide.Amino in the covalent modification sealing polypeptide or carboxyl or this two kinds of groups natural exist in polypeptide and the artificial synthetic polypeptide common.For example the polypeptide aminoterminal residue that is produced in the intestinal bacteria (E.Coli) is at proteolysis first being processed N-formylmethionine always almost.

Modification can be how to produce relevant with this protein.For example, modify by the posttranslational modification ability of host cell and the modification signal deciding in the polypeptid acid sequence for recombinant polypeptide.Therefore when the needs glycosylation, polypeptide normally should be carried out in the eukaryotic cell can carrying out glycosylated host.Insect cell often can carry out the post-translational glycosylation identical with mammalian cell, therefore, has developed the insect cell expression system that can efficiently express the mammalian proteins matter with natural type glycosylation.Similarly thinking also is applicable to other modification.Modification of the same type can occur on the identical or different degree of several site ground in given polypeptide.Given polypeptide can also comprise and surpass one type modification.

" function equivalent " be meant as used herein can show to by in the similar substantially body of the endogenous difference expression gene product of above-mentioned difference expression gene sequence encoding or the protein or the polypeptide of external activity." function equivalent " can also refer in the mode of the corresponding section that is similar to endogenous difference expression gene product substantially and other cell or the outer interaction of molecules of born of the same parents and the protein or the polypeptide of other cell or the outer interaction of molecules of born of the same parents.For example " functional equivalent " peptide can reduce antibody to the combination of endogenous protein corresponding peptides (peptide that promptly has modified aminoacid sequence is to obtain " functional equivalent " peptide) or to the combination of endogenous protein itself, wherein this antibody is at the corresponding peptides generation of this endogenous protein in immune identification method.Peptide etc. the functional equivalent of volumetric molar concentration will reduce in conjunction with aforesaid corresponding peptides at least about 5%, between preferably about 5% to 10%, between more preferably about 10% to 25%, between very preferably about 25% to 50%, and between most preferably about 40% to 50%.

For example the peptide of functional equivalent can possess repertoire and maybe can lack one or more active functions.Therefore variation can influence for example ubiquitin combined function in the present invention, the ubiquitin recognition function, with the interaction of the substrate protein of ubiquitination as combining or proteolyzing, subunit interacts especially in the intravital interaction of proteolytic enzyme, activation or combination by ATP, developmental character is expressed, temporal expression, tissue specific expression, composition such as transcription regulaton factor with cell, especially the interaction of trans-acting transcription regulaton factor, peptide bond in the protease hydrolysis cutting poly ubiquitin and the peptide bond between ubiquitin or poly ubiquitin and the protein substrate, and the peptide bond between protease hydrolysis cutting ubiquitin or poly ubiquitin and peptide or the amino acid.

Functional completely variant generally only comprises conservative property variation or the variation in non-key residue place or non-critical areas.Functional variant also can comprise similar amino acid whose substituting, the described alternative not noticeable change that does not cause the variation of function or cause function.Alternatively, this type of substitute can to function produce to a certain degree actively or negative influence.

The non-functional variant comprises generally that one or more non-conservations amino acid whosely substitute, disappearance, insertion, inversion or brachymemma or substituting in Key residues place or critical area, insertion, inversion, disappearance.Variant such as description can be natural existence or produce by recombinant means or chemosynthesis, with give the ubiquitin protease polypeptide useful with new characteristics.This comprises by stoping protein aggregation to prevent immunogenicity from pharmaceutical preparation.Useful variation further comprises the change of catalytic activity.For example an embodiment relates at the binding site place and causes the variation of peptide bond bonded but not the variation of the peptide bond hydrolysis or the hydrolysis of slowing down.Other useful variation causes the raising of peptide bond hydrolysis speed.The useful variation of another of same loci place can cause the affinity higher or lower to substrate.

Useful variation also comprises the change of the protein substrate avidity that is different from the ubiquitination of normally being discerned.Other the useful variation that relates to the recognition reaction that has changed has influenced the normal substrate type of discerning.For example, but a kind of variation may cause discerning the resistates (remnants) of the intact substrate nonrecognition substrate of ubiquitination, as amino acid of ubiquitination or the peptide as protease hydrolysis effect product, it is the hydrolysis result of the intact substrate of ubiquitination.Perhaps this proteolytic enzyme variation so that it can be able to be discerned one or more residual products and can not be discerned complete protein substrate.Another kind of variation will influence this proteolytic enzyme and save the protein of ubiquitination.Therefore can will be degraded from the protein substrate that protease hydrolysis is saved on usually.More useful variation influences the ability that this proteolytic enzyme is subjected to activator such as cytokine induction, and described activator includes but not limited to the activator that those are disclosed herein.Another useful variation will influence the substrate or the side chain poly ubiquitin substrate of the ubiquitination of poly of poly ubiquitin, the wire of the identification of ubiquitin substrate so that poly ubiquitin that this kind of enzyme can not be discerned one or more wire, side chain.Specific variation comprises that brachymemma for example lacks the HIS structural domain, and this variation has caused taking off the reduction or the forfeiture of ubiquitin action activity.Another kind of useful variation comprises the variation that stops by the activation of ATP.

Another kind of useful variation provides fusion rotein, and merge effectively with from another kind of UBP or from one or more structural domains or the territory, subprovince of UCH in one or more structural domains or territory, subprovince in this fusion rotein.Particularly, can import and give enzyme rescue function that does not normally possess the rescue function or structural domain or the territory, subprovince of discerning the function of specific substrates, wherein original enzyme does not have recognition function.Other variation comprises can be influenced the ubiquitin recognition reaction or discern the variation of the protein substrate of ubiquitination.Other variation can influence specific subunit especially in the intravital interaction of proteolytic enzyme.Other variation will influence developmental character, sequential or tissue-specific expression.Other variation will influence for example interaction between transcription regulaton factor of component in the cell.Amino acid to the function key can be identified by method known in the art, as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham etc., (1985) Science 244:1081-1085).Alanine scanning mutagenesis method each residue place in molecule imports single alanine mutation.Detect the biologic activity of resulting mutating molecule then, as the extracorporeal hydrolysis of peptide or external activity such as proliferation activity, the conduction of receptor-mediated signal and other the cell processes of dependence ubiquitin, include but not limited to those activity relevant disclosed herein with the ubiquitin system.Also can determine for combination or the crucial site of identification, as crystallization, nucleus magnetic resonance or photoaffinity mark (Smith etc. (1992) J.Mol.Biol.224:899-904 by structural analysis; (1992) Science 255:306-312 such as de Vos).

The identification method of taking off the ubiquitin enzymic activity is known for this area crowd and can be at (1997) Journal of Biological Chemistry 272:51-57 such as for example Zhu, Mitch etc. (1999) AmericanJournal of Physiology 276:C1132-C1138, among Liu etc. (1999) the Molecular and CellBiology 19:3029-3038 and (1994) The FASEB Journal 8:182-192 such as those documents in multiple summary, quoted such as Ciechanover, Chiechanover (1994) Biol.Chem.Hoppe-Seyler 375:565-581, Hershko etc. (1998) Annual Reviewof Biochemistry 67:425-479, Swartz (1999) Annual Review of Medicine50:57-74, find among (1998) Critical Reviews in Biochemistry and Molecular Biology33:337-352 such as Ciechanover (1998) EMBO Journal 17:7151-7160 and D ' Andrea.These identification methods include but not limited to the disappearance of substrate, comprise the poly ubiquitin or for example free monomeric appearance of ubiquitin of appearance, gross protein turnover, the specific protein of minimizing, intermediate and the end product of the protein substrate of ubiquitination or protein residue thing quantity turnover, ubiquitin in conjunction with, to the interaction of the combination of the protein substrate of ubiquitination, subunit, with the interaction of ATP, with the stabilization of the interaction of cellular constituent such as trans-acting regulatory factor, specified protein and other.

" host cell " referred to comprise protokaryon or the eukaryotic cell that has imported allogeneic dna sequence DNA by any method as electroporation, calcium phosphate precipitation, microinjection, conversion, virus infection etc. as used herein.

" allogenic " means " belonging to different natural origins " as used herein or represented non-natural state.For example if host cell is with deriving from another kind of biological DNA or the gene transformation that especially derives from another species, this gene is allogenic for the offspring of this host cell and this host cell that has carried this gene.Similarly, allos refers to derive from identical natural, primary cell type and is inserted into the nucleotide sequence of described cell type, but this nucleotide sequence occurs with non-natural state, for example has different copy numbers or is subjected to the control of different controlling elements.

" carrier " is that allogenic nucleic acid can insert nucleic acid molecule wherein, and this subsequently nucleic acid molecule can be directed in the suitable host cell.Carrier preferably has the site that one or more replication orgin and one or more recombinant DNA can insert.Carrier usually has the facilitated method that does not possess the cell of selecting to contain these carriers the cell of these carriers from those, for example these vector encoded drug resistance gene.Common carrier comprises plasmid, viral genome and (mainly in yeast and bacterium) " artificial chromosome ".

" plasmid " usually named herein be small letter p before or after be capitalization and/or numeral, be accustomed to consistent with the standard name that those skilled in the art are familiar with.Initial plasmid disclosed herein or can commercial obtain can openly obtain maybe can make up by the many presentation methods of knowing of routine utilization from obtainable plasmid with nonrestrictive basis.Numerous can be used according to the invention plasmid and other clone and expression vector for well-known and be easy to acquisition into those skilled in the art.In addition, the technician is easy to make up and is applicable to any amount of other plasmid of the present invention.Feature, structure and the purposes of this class plasmid and other carrier will be for technicians because the disclosure will be to understand easily among the present invention.

Term " separated " means and material is removed (for example natural surroundings is naturally occurring as if this material) from the original environment of this material.For example, natural polynucleotide or the polypeptide of existing that is present in the animal alive separates without crossing, but from some of natural system or all the coexisting substances isolating identical polynucleotide or polypeptide be separated, even if this subsequently polynucleotide or polypeptide have imported in this natural system once more.These class polynucleotide can be the part of carrier and/or this class polynucleotide or polypeptide and can be the part of composition, and still are separated, because this carrier and composition are not the part of its natural surroundings.

As used herein, term " transcriptional control sequence " refers to dna sequence dna such as homing sequence, enhancer sequence and promoter sequence, and this dna sequence dna comprises to be induced, suppress or control and the transcribing of the nucleotide sequence of its coded protein that effectively is connected." people's transcriptional control sequence " is naturally to exist in any transcriptional control sequence in the people's gene group and " inhuman transcriptional control sequence " is non-existent any transcriptional control sequence in the people's gene group.

Multiple host-expression vector system can be used for expressing gene coded sequence of the present invention.This class host-expression system is can produce and the carrier of subsequent purificn coding aim sequence is representative, but also be representative with such cell, wherein this cell transform with suitable nucleotide coding sequence or transfection after original position (in situ) show the protein of difference expression gene of the present invention.These host-expression systems include but not limited to recombinant phage dna, plasmid DNA or cosmid DNA expression vector microorganism transformed such as the bacterium (for example intestinal bacteria, subtilis (B.subtilis)) of the protein coding sequence through having comprised the differential expression gene; The yeast (for example yeast saccharomyces cerevisiae (Saccharomyces), pichia spp (Pichia)) that the recombinant yeast expression vector of the protein coding sequence through having comprised the differential expression gene transforms, the insect cell system that infects with the recombinant virus expression vector (for example baculovirus) of the protein coding sequence that contains the differential expression gene; The recombinant virus expression vector of protein coding sequence (cauliflower mosaic virus for example, CaMV through containing the differential expression gene; Tobacco mosaic virus (TMV), TMV) recombinant plasmid transformed carrier (as Ti-plasmids) the plant transformed cell system of infection or protein coding sequence through containing the differential expression gene; Or mammal cell line system (for example COS, CHO, BHK, 293,3T3), this mammal cell line system has carried to contain and has derived from the genomic promotor of mammalian cell (for example metallothionein promoter) or from the promotor of mammalian virus (gland virus stage starting for example; Vaccinia virus 7.5K promotor) recombinant expression construct body.

In bacterial system, numerous expression vectors can advantageously be selected according to the proteinic desired use of expressed differential expression gene.For example when this albumen will produce with generation antibody or screening peptide library in large quantities, may need to instruct high level to be easy to the carrier of the expressing fusion protein of purifying.This class carrier includes but not limited to coli expression carrier pUR278 (Ruther etc., 1983, EMBO is J.2:1791), wherein can individually to be connected to this carrier interior and consistent with lac Z coding region open reading-frame (ORF) so that produce fusion rotein for the protein coding sequence of differential expression gene; PIN carrier (Inouye and Inouye, 1985, Nucleic Acid Res.13:3101-3109; Van Heeke ﹠amp; Schuster, 1989, J.Biol.Chem.264:5503-5509) and other carrier.The PGEX carrier can also be used for expressing as with the allogenic polypeptide of glutathione S-transferase (GST) fusion rotein.Usually this class fusion rotein be soluble and be easy to from through the cracked cell by being adsorbed in glutathione agarose pearl and wash-out and purifying when having free glutathione subsequently.This PGEX carrier design is to have comprised the proteolytic enzyme cutting site of zymoplasm or factor Xa so that can discharge the protein of the target gene through cloning from the GST part.

Promoter region can be selected from the carrier that any required gene uses; wherein said carrier has comprised reporter gene transcriptional units such as chloramphenicol acetyltransferase (" the cat ") transcriptional units that lacks promoter region, and this transcriptional units is positioned at and is used for introducing the segmental restriction site downstream that candidate's promoter fragment promptly comprises promotor.Such as the crowd know that the restriction site that is positioned at the CAT upstream region of gene in carrier is introduced the fragment contain promotor and produced the CAT activity, this CAT activity can detect by the CAT identification method of standard.It is well-known and be easy to obtain to be suitable for the carrier of this purpose.PKK232-8 and pCM7 are two kinds of such carriers.Therefore, the promotor that is used to express polynucleotide of the present invention has not only comprised many promotors of knowing that are easy to obtain but also has comprised the promotor that can utilize reporter gene to be easy to obtain by aforementioned techniques.

The known bacterium promotor that is fit to polynucleotide of the present invention and expression of polypeptides is intestinal bacteria lacI and lacZ promotor, T3 and T7 promotor, T5 tac promotor, λ PR, PL promotor and trp promotor.The known eukaryotic promoter that is fit to this purpose be CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus LTR promotor such as Rous sarcoma virus (" RSV ") LTR promotor and metallothionein promoter such as mouse metallothionein(MT)-I promotor.

In the insect system, autographa california (Autographa californica) nuclear polyhedrosis virus (AcNPV) is one of several insect system as the carrier of expressing alien gene.Virus is grown in greedy noctuid (Spodoptera frugiperda) cell in meadow.The encoding sequence of differential expression gene can be cloned into the nonessential zone (for example polyhedron gene) of virus respectively and place under the control of AcNPV promotor (for example polyhedrin promotor).The encoding sequence that the differential expression gene is inserted in success will cause the inactivation of polyhedron gene and produce the non-build recombinant virus (promptly virus lacks the virus by polyhedron gene encoded protein matter shell) that comprises.These recombinant viruses are used to infect the greedy frugiperda cell in meadow then, express the gene that inserts (for example referring to Smith etc. 1983, J.Virol.46:584, Smith, U.S. Patent number 4,215,051) in these cells.

Numerous expression systems based on virus can use in mammalian host cell.When using adenovirus as expression vector, the control complex body can be transcribed/translate to the purpose encoding sequence of differential expression gene with adenovirus such as late promoter is connected with tripartite leader[.This then mosaic gene can pass through in the body or vitro recombination is inserted the adenoviral gene group.Insertion in this viral genome in the nonessential zone (as area E 1 or E3) has generation vigor and can (for example see Logan and Shenk by the proteinic recombinant virus of differential expression expressing gene in the host who has infected, 1984, Proc.Natl.Acad.Sci.USA 81:3655-3659).Also may need to be used for the specificity start signal of the efficient translation of differential expression gene coded sequence of being inserted.These signals comprise ATG initiator codon and the sequence of closing on.When the initiator codon that comprises gene self is inserted into suitable expression vector with the complete differential expression gene that closes on sequence, can no longer need extra translation control signal.Yet when only the encoding sequence of the differential expression gene of part inserts, must provide the translation control signal of external source, may comprise the ATG initiator codon.And this initiator codon must be confined mutually (in phase) to guarantee the translation of whole insets with the purpose reading.The translation control signal and the initiator codon of these external sources can be multiple source, promptly can be the natural synthetic that also can be.The efficient of expressing can (be seen Bittner etc. 1987, Methods in Enzymol by comprising that suitable transcriptional enhancer element, transcription terminator or the like strengthen.153：516-544)。

The selection that is used for suitable carrier that host cell expresses and promotor is the method known of crowd and is used for expression vector establishment, carrier imports in the host cell and expresses required technology itself in the host is exactly routine techniques in this area.

Generally, recombinant expression vector comprise replication orgin, derived from cance high-expression gene so that instruct promotor that the downstream configurations sequence transcribes to be exposed to the cell that contains this carrier behind this carrier so that separate with selected marker.

Can select to regulate the expression of institute's insertion sequence in addition or modify and the host cell strain of processed gene product with needed ad hoc fashion.This type of modification of protein (for example glycosylation) and processing (for example cutting) may be important to proteinic function.Different host cells have characteristic and particular proteins translation post-treatment and modified mechanism.Can select suitable clone or host system to guarantee the correct modification and the processing of expressed exogenous protein.For this purpose, can use have be used for that primary transcript is correctly processed, the eukaryotic host cell of the cell mechanism of gene product glycosylation and phosphorylation.This type of mammalian host cell includes but not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293,3T3, WI38 etc.

In order to produce recombinant protein for a long time in large quantities, preferred stably express.But the proteinic clone of design stability differential expression expressing gene for example.The DNA of the expression controlling elements that host cell can be fit to (for example promotor, enhanser, sequence, transcription terminator, polyadenylation site etc.) and selected marker control but not the expression vector that comprises the virus replication starting point transform.After importing foreign DNA, the cell of through engineering approaches can be grown in rich medium 1-2 days, transferred selective medium then to.Thereby the selected marker in the recombinant plasmid provides resistance for chosen process and make cell this plasmid of stable integration in the karyomit(e) of cell make the cell growth form transforming focus, and next this transforming focus can and expand to clone through the clone again.Can advantageously make in this way protein expression clone with artificial poor designs opposite sex expressing gene.The clone of this through engineering approaches especially influences aspect the active compound of proteinic endogenous of differential expression gene useful in screening and assessment.

Can use the multiple choices system, include but not limited to herpes simplex virus thymidine kinase (Wigler etc., 1977, Cell 11:223), hypoxanthine guanine phosphoribosyltransferase (Szybalska and Szybalski, 1962, Proc.Natl.Acad.Sci.USA 48:2026), and adenine phosphoribosyltransferase (Lowy etc., 1980, Cell 22:817) gene can be applied to tk respectively ^-, hgprt ^-Or aprt ^-Cell.The metabolic antagonist resistance can be used as the basis of selecting dhfr, gpt, neo and hygro in addition, and wherein said dhfr provides methotrexate resistance (Wigler etc., 1980, Natl.Acad.Sci.USA77:3567; O ' Hare etc., 1981, Proc.Natl.Acad.Sci.USA 78:1527); Gpt provides mycophenolic acid resistance (Mulligan ﹠amp; Berg, 1981, Proc.Natl.Acad.Sci.USA 78:2072); Neo provide aminoglycoside G-418 resistance (Colberre-Garapin etc., 1981, J.Mol.Biol.150:1); And hygro provide hygromycin resistance (Santerre etc., 1984, Gene30:147).

Another kind of alternative fusion rotein system can make the non-sex change fusion rotein of expressing among the human cell line be easy to purifying (Janknecht etc., 1991, Proc.Natl.Acad.Sci.USA 88:8972-8976).In this system, with the goal gene subclone to vaccinia recombination plasmid so that by translation open reading-frame (ORF) that makes this gene and the aminoterminal label fusion of forming by six histidine residues.The extract that derives from the cell that infects vaccinia virus recombinant is uploaded to Ni ²⁺Nitrilo acetic acid-agarose column has added histidine-tagged protein with the damping fluid selective elution that contains imidazoles subsequently.

When as the component in the identification method as described below system, the protein of differential expression gene can through direct or indirect mark with convenient by the differential expression gene protein and the test substrate between the detection of the complex body that formed.Can use in the multiple suitable Mk system any one, include but not limited to radio isotope as ¹²⁵I; The enzyme labelling system, wherein this kind of enzyme Mk system produces detectable calorimetric signal or light when being exposed to substrate; And fluorescent marker.

When using recombinant DNA technology to be used for the protein of differential expression gene of this class identification method system with generation, advantageously fusion rotein is designed to be convenient to mark, fixing and/or detect.

Indirect labelling relates to protein as the use of traget antibody, and its specificity is bonded to the product of differential expression gene.This antibody-like includes but not limited to antibody polyclonal, monoclonal, chimeric, strand, Fab fragment and the fragment that is produced by the Fab expression library.

Another aspect, the polypeptide of ubiquitin specific protease of the present invention is useful in the biological assay relevant with the ubiquitin protease function.This class identification method comprises any known function or any known activity or the characteristic useful to diagnosis and the treatment disease relevant with ubiquitin or ubiquitin proteolytic enzyme.Potential identification method discloses and generally includes substrate disappearance, end product appearance and gross protein or specified protein turnover at this.

Ubiquitin specific proteins enzyme polypeptide also is used for the identification method based on cell or the screening of cell free system Chinese traditional medicine.Based on the system of cell can be natural system as the cell as the examination of living tissue or the ubiquitin proteolytic enzyme normal expression of breeding in cell cultures.Yet in one embodiment, the identification method based on cell relates to the recombinant host cell of expressing ubiquitin proteolytic enzyme.Determination test compound and the interactional ability of ubiquitin proteolytic enzyme also can comprise test compound when measuring the ability comparison that combines polypeptide with known binding molecule (as ubiquitin) preferentially in conjunction with as described in the ability of polypeptide.Described polypeptide can be used for identifying the compound of regulating the ubiquitin protease activity.This compounds for example can improve or reduce poly ubiquitin to wire or side chain, the protein substrate or the affinity of the resistates of the protein substrate of ubiquitination of ubiquitination.This compounds also can for example improve or reduce and the association rate of these components.This compounds can also with these components competition combining or substituting these and be bonded to the component of ubiquitin proteolytic enzyme to ubiquitin proteolytic enzyme.This compounds can also influence with the interaction of other component as with ATP, 19S mixture in other subunit and the interaction of transcription regulaton factor.Therefore be to be understood that this compounds not only can be by ubiquitin but also can by with any component evaluation of disclosed proteolytic enzyme generation functional interaction.This includes but not limited to arbitrary these components disclosed herein.

Ubiquitin specific protease, derivative and fragment can be used for high flux screening so that identify the ability of candidate compound in conjunction with ubiquitin proteolytic enzyme.These compounds can further screen with functional ubiquitin proteolytic enzyme so that measure the influence of compound to the ubiquitin protease activity.Can identify and make ubiquitin proteinase activated (agonist) or inactivation (antagonist) compound to the purpose degree.Control method can externally implement to implement in (for example by this reagent is cultivated with cell) or the body (for example by use this reagent to the experimenter).

Ubiquitin specific proteins enzyme polypeptide of the present invention can be used in that SCREENED COMPOUND stimulates or suppresses the ubiquitin protease protein and the interaction between the target molecule of ubiquitin protease protein of interacting usually.Target molecule can be ubiquitin, the substrate of ubiquitination or another component (for example ATP) in poly ubiquitin or the ubiquitin protease protein normal phase mutual effect approach.Identification method comprises step: ubiquitin protease protein and candidate compound make up under permission ubiquitin protease protein or fragment and the interactional condition of target molecules; And the biological chemistry consequence that detects formation or the detection ubiquitin proteolytic enzyme and the target molecules interphase interaction of complex body between ubiquitin protease protein and the target molecules.Can identify any activity relevant with protease function.This comprises the peptide substrates that produces hydrolysate such as free-end, the hang oneself free-end amino acid of hydrolysis substrate, the free ubiquitin, low molecular weight substance through the poly ubiquitin of hydrolysis, owing to save the intact substrate protein that is discharged from the proteolysis effect, the free poly ubiquitin that hydrolysis poly ubiquitin forms from intact substrate, and amino acid and the peptide of substrate resistates as producing from the biology terminal point of proteolysis effect of substrate protein white matter or ubiquitin approach.

Mensuration ubiquitin proteolytic enzyme is bonded to the ability of target molecules also can finish (Sjolander etc. by using as the technology (BIA) of real-time bimolecular transactional analysis method, (1991) Anal.Chem.63:2338-2345 and Szabo etc., (1995) Curr.Opin.Struct.Biol.5:699-705).As used herein, " BIA " is a kind of technology (as BIAcore ) that real-time biologic specificity interacts and needn't any interactant of mark that is used to study.Optical phenomena in the surface kytoplasm resonance (SPR) changes the real time reaction that can be used to indicate between the biomolecules.Test compound of the present invention can use arbitrary method of the several different methods in the combinatorial library method known in the art to obtain, and comprises biological library; Addressable parallel solid phase in space or liquid phase library; Need go the synthetic library method of flatung; " pearl one compound " library method; And the synthetic library method of using affinity chromatography to select.Biological library method is limited to polypeptide libraries and other four kinds of methods are applicable to the small molecules library (Lam, K.S. (1997) Anticancer Drug Des.12:145) of polypeptide, non-peptide oligomer or compound.

The example of the method in synthetic molecules library can find in the art, as at DeWitt etc., (1993) Proc.Natl.Acad.Sci.USA 90:6909; Erb etc., (1994) Proc.Natl.Acad.Sci.USA 91:11422; Zuckermann etc., (1994) .J.Med.Chem.37:2678; Cho etc., (1993) Science 261:1303; Carell etc., (1994) Angew.Chem.Int.Ed.Engl.33:2059; Carell etc., (1994) Angew.Chem.Int.Ed.Engl.33:2061; And, find among (1994) J.Med.Chem.37:1233 at Gallop etc.Can there be (for example Houghten (1992) Biotechniques13:412-421) in the library of compound or (Lam (1991) Nature 354:82-84), chip (Fodor (1993) Nature 364:555-556), bacterium (Ladner U.S. Patent number 5 on pearl in solution, 223,409), spore (Ladner U.S. Patent number 5,223,409), plasmid (Cull etc., (1992) Proc.Natl.Acad.Sci.USA89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla etc., (1990) Proc.Natl.Acad.Sci.97:6378-6382); (Felici (1991) J.Mol.Biol.222:301-310); (Ladner supra) presents.Candidate compound for example comprises 1) peptide such as soluble peptide, comprise that afterbody connects the member of fusogenic peptide and the random peptide library of Ig and (for example sees Lam etc., (1991) Nature 354:82-84; Houghten etc., (1991) Nature 354:84-86) and the derivative molecular library of forming by D-and/or L-conformation amino acid of combinatorial chemistry; 2) the phosphinylidyne peptide (for example at random with the member of part degeneracy, directed phosphinylidyne peptide library, see for example Songyang etc., (1993) Cell 72:767-778); 3) antibody (for example polyclonal, monoclonal, humanization, antiidiotype, chimeric and single-chain antibody and Fab, F (ab ') ₂, Fab expression library fragment and antibody the epi-position binding fragment); And 4) little organic and inorganic molecule (for example molecule that from combination and library natural product, obtains).

A kind of candidate compound is total length solubility ubiquitin specific protease or its fragment that can compete bound substrates.Other candidate compound comprises ubiquitin protease mutant or its respective segments, and wherein said ubiquitin protease mutant or its respective segments contain the function of influential ubiquitin proteolytic enzyme and to the sudden change of substrate competition.Therefore the present invention for example comprises with than the fragment of high-affinity competition substrate or combine substrate but the fragment of hydrolysising peptide key not.But other candidate compound comprises in conjunction with this proteolytic enzyme does not discharge or discharge slowly the protein of ubiquitination or protein analogue.Other candidate compound comprises the analogue of other natural substrate, as in conjunction with but be not released or discharge substrate resistates slowly.Other candidate compound comprises the activator such as the cytokine of proteolytic enzyme, includes but not limited to disclosed herein those.

The invention provides other terminal point of identifying the compound of regulating (stimulating or inhibition) ubiquitin protease activity.This class identification method relates generally to the evaluation of the incident of indication ubiquitin protease activity in the approach.This can comprise coming the cell incident of autospasy ubiquitin effect, for example signal conduction or arbitrary cell processes (including but not limited to the cell processes that comes the effect of autospasy ubiquitin that those are disclosed herein) of cell cycle progression, apoptosis, somatomedin mediation.Specific phenotype is included in the reverse of stress reaction, dna replication dna, acceptor internalization, cell transformation or conversion and the variation of Transcriptional Silencing aspect.These identification methods are based on the various kinds of cell sexual function of taking off the ubiquitin enzyme.These enzymes act on the adjusting of protein ubiquitination on a plurality of different levelss.

Taking off the ubiquitin enzyme can be monomer ubiquitin molecule with the poly ubiquitin chain degradation of wire.Taking off ubiquitin enzyme such as isopeptidase T can make the dendritic multipass of branch be degraded to monomeric ubiquitin molecule at protein chain.Take off the ubiquitin enzyme and can from the target proteins matter that is conjugated with ubiquitin, remove ubiquitin.Taking off ubiquitin enzyme such as FAF or PA700 isopeptidase can remove the poly ubiquitin and therefore save target from the degraded of 26S proteasome from the target proteins matter of ubiquitination.Take off ubiquitin enzyme such as Doa-4 and can from the proteasome degraded product, remove the poly ubiquitin.The result of all these effects is that the ubiquitin storehouse of cell free monomers is regulated.Therefore, identification method can be based on detecting through hydrolysis/take off any product that the ubiquitin effect is produced.Further can identify the expression of gene that is subjected to the effect of ubiquitin proteolytic enzyme and raises or reduce.In one embodiment, the regulation domain of this genoid can be connected to the mark such as the luciferase of easy detection effectively.Therefore any biology or biochemical function by the mediation of ubiquitin proteolytic enzyme can be used as the terminal point identification method.That these functions comprise is described herein, quote as a reference, with reference to included all biological/biological chemistry incident, and be other functions known to those of ordinary skills as these terminal point identification method targets.

In conjunction with and/or activated compounds also can be by using chimeric ubiquitin protease protein screening, wherein so the place disclose in the described chimeric ubiquitin protease protein one or more structural domains, site etc. or their part can be by the allos counterpart replacement that is derived from other ubiquitin proteolytic enzyme.Can use for example differing substrate specificity and/or interactional identification of affinity or calmodulin binding domain CaM to be different from natural ubiquitin proteolytic enzyme.Therefore the different pathway component of a cover can be used as activated terminal point identification method.Further, being responsible for developmental character, sequential or tissue-specific site can be replaced so that this proteolytic enzyme can detect under the condition that special developmental character, sequential or the sense of organization are expressed by the allos site.

The ubiquitin protease polypeptide also is used for competitive in conjunction with identification method, in design is used for finding method with the interactional compound of ubiquitin proteolytic enzyme.Therefore compound this compound combine with the ubiquitin protease polypeptide or interactional condition under be exposed to the ubiquitin protease polypeptide.Solubility ubiquitin protease polypeptide also can add in this mixture.If test compound and this solubility ubiquitin protease polypeptide interact, then this compound has reduced the amount of formed complex body or the activity of ubiquitin proteolytic enzyme target.The identification method of this type is particularly useful for seeking and the interactional compound in ubiquitin proteolytic enzyme specific region.Therefore be designed to comprise peptide sequence with the soluble polypeptide of ubiquitin proteolytic enzyme target region-competitive corresponding to the purpose zone.

The competition of another kind of type can be used for finding and the specific interactional compound in functional site in conjunction with identification method.For example, ubiquitin and candidate compound can be added to the sample of ubiquitin proteolytic enzyme.And ubiquitin proteolytic enzyme interacts will reduce the amount of formed complex body between ubiquitin proteolytic enzyme and the ubiquitin in the compound in the same site of ubiquitin.Therefore might find that specificity stops the compound of ubiquitin proteolytic enzyme and ubiquitin interphase interaction.

Another example has related in the sample of ubiquitin proteolytic enzyme and poly ubiquitin and has added candidate compound.Amount or poly ubiquitin the combining that to reduce hydrolysis with the compound of poly ubiquitin competition to ubiquitin proteolytic enzyme.Therefore the compound that can find directly to interact and compete with the poly ubiquitin with ubiquitin proteolytic enzyme.This class identification method can relate to and interactional any other component of ubiquitin proteolytic enzyme, for example the substrate protein white matter of ubiquitination, ubiquitination the substrate resistates and with interactional cellular component of this proteolytic enzyme such as transcription regulaton factor.For implementing acellular drug screening identification method, wish fixedly ubiquitin proteolytic enzyme or its fragment or fix its target molecules and from one or both proteinic non-complexed forms formulas, separate with convenient mixture, and the automatization of adaptation identification method.The technology of fixing protein can be used for the drug screening identification method on matrix.

In one embodiment, can provide fusion rotein, this fusion rotein has added so that make the structural domain of this protein bound matrix.For example glutathione-S-transferase/ubiquitin proteolytic enzyme fusion rotein is adsorbable in glutathione agarose pearl (Sigma Chemical, St.Louis, Mo.) or gsh deutero-microtiter plate, this subsequently fusion rotein and cell lysate (for example through 35S-mark) and candidate compound mixes and mixture is being beneficial to condition (for example salt of physiological condition and the pH) incubation that mixture forms.Behind the incubation with the washing of this sepharose 4B to remove any unconjugated marker and matrix to be fixed and radioactively labelled substance is directly measured or measure in supernatant from the mixture back of dissociating.

Alternatively, mixture can dissociate from matrix, separates and the standard of use electrophoretic technique quantitatively is found in ubiquitin proteolytic enzyme-conjugated protein level in the sepharose 4B from gel by SDS-PAGE.For example the target molecules of polypeptide or this peptide can utilize the technology of using this area crowd to know of puting together of vitamin H and streptavidin to fix.Alternatively, with proteins react but do not disturb the antibody of this protein bound target molecules to derive to plate well, and this protein can be caught by the antibody coupling effect in the hole.In conjunction with the target composition prepared product of ubiquitin proteolytic enzyme such as ubiquitin, poly ubiquitin, ubiquitination protein substrate, ubiquitination the protein substrate resistates or the amino acid of ubiquitination resistates and candidate compound in the hole that has ubiquitin proteolytic enzyme incubation and can dosing hole in the amount of the mixture of being caught.

Detect the method for this class mixture, except those are used for fixing the method for GST mixture as mentioned above, also comprise the immunodetection of using antibody complex, wherein antibody and ubiquitin proteolytic enzyme target molecules are reacted or are competed with the ubiquitin mmp reaction and with target molecules; And rely on the enzyme connection identification method detect the relevant enzymic activity of target molecules therewith.

The instrumentality of the ubiquitin protease activity of being identified according to these drug screening identification methods can be by handling the experimenter that the cell of expressing ubiquitin proteolytic enzyme be used for the treatment of the disease of suffering from the mediation of ubiquitin proteolytic enzyme approach.

In one aspect, the present invention relates to the instrumentality of genetic expression, wherein said gene comprises the nucleotide sequence shown in SEQ ID No.2, SEQ ID No.6 or SEQ.ID No.10.This class instrumentality preferably inhibition nucleic acid such as antisense oligonucleotide, triplet helical dna, siRNA, ribozyme, RNA is fit (aptamer) or the RNA of two strands or strand.The nucleic acid of design inhibition of gene expression is well known by persons skilled in the art, and wherein said gene comprises the nucleotide sequence shown in SEQ ID No.2, SEQ ID No.6 or SEQ.ID No.10.This inhibition nucleic acid is siRNA in an especially preferred embodiment.Preferred siRNA molecular length is generally between 18 to 30 Nucleotide, though longer length also is suitable for suppressing expression of target gene.In a preferred embodiment, the length of siRNA molecule is between 19 to 25 Nucleotide.

According to the present invention, found the polypeptide that contains the aminoacid sequence that is selected from SEQID No.1, SEQ ID No.5 or SEQ.ID No.9 that in breast cancer tissue, exists content to raise.Therefore the present invention provides the method that is used for the treatment of mammary cancer in one aspect, comprise the inhibition nucleic acid of granting significant quantity to the patient with breast cancer, wherein this inhibition nucleic acid is suitable for suppressing to contain the expression of gene that is selected from nucleotide sequence shown in SEQ ID No.2, SEQID No.6 or the SEQ.ID No.10.In a preferred embodiment, inhibition nucleic acid is siRNA.In another preferred embodiment, nucleotide sequence is SEQ.ID.No.2.In another embodiment, the invention provides the purposes of inhibition nucleic acid, preferably provide the purposes of siRNA, to be used to prepare the medicine for the treatment of mammary cancer, wherein said inhibition nucleic acid is suitable for suppressing to contain the expression of gene that is selected from nucleotide sequence shown in SEQ ID No.2, SEQ ID No.6 or the SEQ.ID No.10, the preferred expression of gene that suppresses to contain nucleotide sequence shown in the SEQ ID No.2.

According to the present invention, found the polypeptide that contains the aminoacid sequence that is selected from SEQ ID No.1, SEQ ID No.5 or SEQ.ID No.9 that especially in lymphoidocyte, exists content to raise in peripheral blood cells.Therefore the present invention provides in one aspect and has been used for the treatment of leukemic method, comprise inhibition nucleic acid from significant quantity to the leukaemic that use, wherein said inhibition nucleic acid is suitable for suppressing to contain the expression of gene that is selected from nucleotide sequence shown in SEQ ID No.2, SEQ ID No.6 or the SEQ.ID No.10.In a preferred embodiment, this inhibition nucleic acid is siRNA.In another preferred embodiment, nucleotide sequence is SEQ.ID.No.6.In another embodiment, the invention provides the purposes of inhibition nucleic acid, preferably provide the purposes of siRNA, be used for the leukemic medicine of preparation treatment, wherein said inhibition nucleic acid is suitable for suppressing to contain the expression of gene that is selected from nucleotide sequence shown in SEQ ID No.2, SEQ IDNo.6 or the SEQ.ID No.10, the preferred expression of gene that suppresses to contain nucleotide sequence shown in the SEQ IDNo.6.

According to a further aspect in the invention, provide and contained the especially pharmaceutical composition of siRNA and pharmaceutically acceptable carrier of inhibition nucleic acid, wherein said inhibition nucleic acid is suitable for suppressing to contain the expression of gene that is selected from nucleotide sequence shown in SEQ IDNo.2, SEQ ID No.6 or the SEQ.ID No.10.

In one aspect of the invention, the diagnostic method of the disease that relates to the ubiquitin specific protease is provided, this method is included in the amount of measuring polynucleotide of the present invention or polypeptide in the people source tissue, and the amount of wherein said polynucleotide or polypeptide has diagnostic significance with respect to the amount rising of this Nucleotide or polypeptide in the normal control tissue to suffering from the human diseases relevant with the ubiquitin specific protease.Term " normally " means the human tissue that derives from the disease to be diagnosed of trouble not in the context of the used tissue of diagnostic method of the present invention.In the embodiment preferred in this regard, these class methods are implemented at external or stripped (ex vivo).

Of the present invention one preferred aspect, be used to diagnose the method for the disease that relates to the ubiquitin specific protease to comprise and relate to the detection step that antibody is contacted with tissue, wherein said antibodies specific is in conjunction with the polypeptide that contains aminoacid sequence shown in arbitrary SEQ ID No.1, SEQ ID No.5 or the SEQ.ID No.9, and detect described antibody and combine with the specificity of polypeptide in described tissue, wherein the detection of specificity bonded polypeptide has been indicated to have the polypeptide that contains aminoacid sequence shown in arbitrary SEQ ID No.1, SEQ ID No.5 or the SEQ.ID No.9.

In one embodiment of the invention, the cell of being diagnosed be mammary gland cell and related disease include but not limited to breast development disorder, inflammation include but not limited to acute mastitis, periductal mastitis (property retransmitted subareolar abscess, lactiferous duct squamous metaplasia), mammary duct ectasia, steatonecrosis, granuloma mazoitis with the relevant pathology of siloxanes breast implant; The fibrous capsule variation; The proliferative cystic hyperplasia of breast includes but not limited to epithelial hyperplasia, sclerosing adenosis and ductule papilloma; Tumour includes but not limited to mesenchymoma for example fibroadenoma, phyllodes tumor and interior knurl, and epithelioma such as big duct papilloma; Mastocarcinoma comprises that carcinoma in situ (Non-Invasive) comprises DCIS (comprising Paget ' s disease) and LCIS and invasive carcinoma (infiltration), includes but not limited to non-specific infitrating ductal carcinoma, infiltrating lobular carcinoma, medullary carcinoma, colloid (mucus) cancer, tubular carcinoma and wetting property papillary carcinoma and multiple malignant tumour.The male breast disease includes but not limited to gynecomastia and cancer.

In another embodiment of the invention kind, the cell of being diagnosed is a peripheral blood cells, especially lymphoidocyte.Disease includes but not limited to leukemia.

Again in another embodiment of the invention, the cell of being diagnosed is positioned at brain and participates in disease of brain, and described disease of brain includes but not limited to relate to neuronic disease and relates to for example disease of stellate cell, oligodendrocyte, ependymocyte and microglia of neuroglia; Cerebral edema, intracranial pressure raises and herniae, and hydrocephalus; Deformity and developmental character disease such as neural tube defect, preceding abnormalities of brain, postfovea unusual and syringomyelia and hydromyelia; Perinatal period brain injury; Cerebrovascular disease disease such as those and relevant cerebrovascular disease anoxic, local asphyxia and infraction comprise infraction that ypotension, hypoperfusion and low flow state-whole cerebral ischemia and local cerebral ischemia-derive from local blood supply blocks, intracranialing hemorrhage comprises that ICH, subarachnoid hemorrhage and berry aneurysm break and vascular malformation, the hypertensive cerebral cardiovascular and cerebrovascular diseases comprises lacunar infarction, the hemorrhage and hypertensive encephalopathy in slit; Infect as lopsided meningitis and comprise acute festering type (bacillary) meningitis and polarity sterility (viral) meningitis, acute focal pyogenic infection comprises cerebral abscess, subdural empyema and epidural abscess, the chronic bacillary meningoencephalitis comprises the sick and branch mycobacterium bacterium of tubercule bacillus, neurosyphilis and neural Greensboro are dredged sick (Lyme disease), viral meningoencephalitis comprises arthropod-borne (Arbo) viral encephalitis, herpes simplex virus type 1, herpes simplex virus type 2, varicella zoster virus (Herpes zoster), cytomegalovirus, poliomyelitis, rabies, comprise HIV-1 meningoencephalitis (subacute encephalitis) with the human immunodeficiency virus I, the cavity myelopathy, the myopathy that AIDS is relevant, peripheral neurophaty and children AIDS, many kitchen ranges of carrying out property eukoencephalopathy, subacute sclerosing panencephalitis, the fungoid meningoencephalitis, neural other infectious diseases; Transmissible spongiform encephalopathy (prion disease); The disease of demyelination comprises that multiple sclerosis, multiple sclerosis variant, acute disseminating property encephalomyelitis and acute necrotizing hemorrhagic encephalomyelitis and other have the disease of demyelinization; Degenerative disease is as influencing the degenerative disease that corticocerebral degenerative disease comprises alzheimer's disease and Pick disease, basal nuclei and brain stem, comprises that parkinson's syndrome, primary parkinson's syndrome (Parkinsonism), stein-leventhal syndrome, the sex change of cortex oblongata, multiple system atrophy comprise that line deceives mutagenicity, summer moral syndrome and olivopontocerebellar atrophy and Huntington Chorea; Spinocerebellar degeneration comprises spinocerebellar ataxia, comprise that Freed relies western ataxia and ataxia ataxia-telanglectasia, the degenerative disease that influences motor neuron comprises amyotrophic lateral sclerosis (motor neuron), oblongata myelatrophy (Kennedy Foster Kennedy syndrome) and Duchenne-Arandisease; Inborn errors of metabolism such as alba under-nutrition, comprise Krabbe disease, metachromatic leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease and canavanine enzymophathy, mitochondrial encephalomyopathy comprises Leigh and other mitochondrial encephalomyopathy; Toxic and acquired metabolic trouble, comprise the not enough and vitamin B12 deficiency of vitamin deficiency VitB1 (VITMAIN B1), neural sequela due to the metabolism disorder, comprise hypoglycemia, hyperglycemia and hepatogenic encephalopathy, toxic disorder comprises carbon monoxide, methyl alcohol, ethanol and radiation, comprises the damage of associativity methotrexate and radioactivity inductive; Tumour such as neurospongioma, comprise astrocytoma, comprise fibril (dispersivity) astrocytoma and glioblastoma multiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, with the brain stem neurospongioma, oligodendroglioma, with ependymoma and the relevant other a large amount of damages in chamber, the neurone tumour, PD tumour comprises medulloblastoma, other parenchymal tumor, comprise elementary brain lymphoma, gonioma, with the pineal gland achiblastoma, durosarcoma, metastatic tumo(u)r, paraneoplastic syndrome, peripheral nerve sheath knurl comprises schwannoma, neurofibroma and pernicious peripheral nerve sheath knurl (malignant schwannoma), and neurocutaneous syndrome (purplish or white patches on the skin mole disease), comprise neurofibromyxoma, it comprises neurofibromatosis 1 type (NF1) and neurofibromatosis 1 type (NF2), tuberous sclerosis and Von Hippel-Lindau disease.

The purposes that the invention still further relates to polypeptide of the present invention be provided for diagnosing the illness or to the ubiquitin specific protease the target of disease mediated susceptibility, wherein said disease includes but not limited to relate to the disease of the tissue of expressing ubiquitin proteolytic enzyme as disclosed here, for example mammary cancer.Thus, provide and be used for detecting cell, tissue or the existence of organism ubiquitin proteolytic enzyme or the method for level.Described method comprises makes the biological sample contact can be with the interactional compound of ubiquitin proteolytic enzyme so that can detect this interaction.Described polypeptide also is used for the treatment of the disease that is reduced to feature with the amount of this constituents.Therefore, it is useful to treating to increase or reduce this protease activities.Described polypeptide is also diagnosed the disease that is reduced to feature with too much substrate or substrate level to providing target to be used to.Under the too much situation of substrate, use the polypeptide of this proteolytic enzyme that a kind of diagnostic identification method can be provided thus.

And for example have and reduced active proteolytic enzyme and can be used for diagnosing the illness, wherein the minimizing of substrate is the reason of this disease.A kind of reagent that is used to detect ubiquitin proteolytic enzyme be can selective binding ubiquitin proteolytic enzyme antibody.Biological sample comprises from the isolating tissue of experimenter, cell and biological fluid and the tissue that exists in subject, cell and body fluid.Ubiquitin proteolytic enzyme also provides target to be used to diagnose active disease or has had the susceptibility of the patient of ubiquitin ease variants to disease.Therefore ubiquitin proteolytic enzyme can separate from biological specimen and identify whether there is the heredity sudden change that causes abnormal protein.Described sudden change comprises amino acid replacement, disappearance, insertion, rearrangement (as the result of aberrant splicing incident) and inappropriate posttranslational modification.Analytical procedure comprises the electrophoretic migration of change, trypsinase is to the change of peptide digestion, the ubiquitin protease activity that in based on cell or acellular identification method, changes, in conjunction with or the change of hydrolysis poly ubiquitin, in conjunction with the substrate protein white matter of ubiquitination or the change of hydrolysis ubiquitin from this protein, comprise peptide or amino acid and the change of hydrolysis ubiquitin from this class resistates in conjunction with the protein residue thing of ubiquitination, the change of total protein turnover, the change of specified protein turnover, the change of antibodies mode, the iso-electric point that changes, directly order-checking amino acid and arbitrary other are used for protein prevailingly or are used for the known authenticate technology that ubiquitin proteolytic enzyme detects sudden change specifically, comprise the identification method that discuss in this place.The ex vivo technique that is used to detect ubiquitin proteolytic enzyme comprises enzyme linked immunological identification method (ELISA), western blotting, immunoprecipitation and immunofluorescence.

Alternatively, can be by import this protein that detects in the body through the anti-ubiquitin protease antibody of mark among the experimenter to the experimenter.For example antibody can detect the existence and the position of this radioactivity sign among the experimenter by the standard imaging technique with radioactivity sign mark in addition.What be particularly useful is to detect the segmental method of ubiquitin proteolytic enzyme in the method for the ubiquitin proteolytic enzyme allelic variant that the experimenter expresses and the test sample.The ubiquitin protease polypeptide also is used for the pharmacogenomics analysis.Pharmacogenomics relates to the clinical significant inheritable variation of drug reaction, and this inheritable variation is attributed to the change or the unusual effect of patient's Chinese traditional medicine tendency of getting involved.Referring to for example Eichelbaum, M. (1996) Clin.Exp.Pharmacol.Physiol.23 (10-11): 983-985 and Linder, M.W. (1997) Clin.Chem.43 (2): 254-266.The clinical effectiveness of these variations is because metabolic individuality difference causes the serious toxicity of medicine in some individuality or the failure of some individual Chinese traditional medicine treatment.Therefore individual genotype can determine therapeutic compound to the mode of action of body or the mode of this compound of organism metabolism.And the activity of drug metabolism enzyme had both influenced pharmaceutically-active intensity and had also influenced the pharmaceutically-active extended period.Therefore individual pharmacogenomics allows to select compounds effective and this compounds to be used for significant quantity based on Id preventative or therapeutic treatment.The discovery of genetic polymorphism in the some drugs metabolic enzyme explained the drug effect why some patient does not obtain to expect through use standard drug dosage, shown the over-drastic drug effect or experienced serious poisoning.Polymorphism can extensive metabolizer's phenotype or the statement of poor metabolizer's phenotype.Therefore, genetic polymorphism can produce ubiquitin proteolytic enzyme equipotential protein variant, and one or more ubiquitin protease functions of described protein variant are different from the corresponding function of protein variant described in another colony in a colony.Therefore described polypeptide allows target to determine to influence the heredity tendency of therapeutic modality.Therefore in the treatment based on ubiquitin, polymorphism can produce more active or more sluggish catalysis region.Therefore, dosage is carried out necessary adjustment so that the result of treatment among the given crowd who has polymorphism is maximized.As genotypic alternative, can identify specific polymorphism polypeptide.

The ubiquitin protease polypeptide also is used for clinical trial and other treatment monitoring therapeuticing effect.Therefore can during whole use is as the treatment of the ubiquitin protease polypeptide of terminal point target, monitor the designed result of treatment that is used to increase or reduce the reagent of genetic expression, protein level or ubiquitin protease activity.Monitoring can followingly be carried out: the sample before (i) obtaining to use before agent administration from the experimenter; (ii) detect and use protein expression level or activity in the preceding sample; (iii) from this experimenter, obtain the sample after one or more the using; (iv) detect and use protein expression level or activity in the sample of back; (protein expression level or active and use protein expression level and activity described in the sample of back in the sample before v) relatively using; And (vi) increase or reduce using of this reagent in view of the above.

In another aspect of this invention, the method that is used for the treatment of includes but not limited to utilize competition to comprise the fragment of the solubility ubiquitin specific protease or the ubiquitin specific protease of the substrate that those are disclosed herein.This class ubiquitin proteolytic enzyme or fragment can have the avidity higher to target, thereby effective competition effect is provided.When improper underground accent and/or when increasing this protein active and can produce useful the influence, active stimulation is required.Similarly, when improper underground accent and/or when increasing this protein active and can produce useful the influence, active inhibition is required.In the example of a this situation, the patient suffers from the disease that is divided into feature with anormogenesis or abnormal cells.In another example, the patient suffers from proliferative disease (as cancer) or is the disease of feature with the abnormal hematopoiesis reaction.In another example, finishing of tissue regeneration is required (is needed as having suffered brain or Spinal injury the experimenter and having organized with the mode regenerating nerve unit that is regulated) among the experimenter.

In another aspect of this invention, provide the method that is used to produce antibody, but one or more epi-positions of wherein said antibody recognition differential opposite sex expressing gene.This antibody-like can include but not limited to polyclonal antibody, monoclonal antibody (mAbs), humanization or chimeric antibody, single-chain antibody, Fab fragment, F (ab ') ₂The epi-position binding fragment of fragment, the fragment that is produced by the Fab expression library, anti-unique sexual type (anti-Id) antibody and any above-mentioned antibody.This antibody-like can be used for for example detecting the gene in finger printing, target, the biological sample or is used as the active instrument of inhibition aberrant gene.

For producing antibody at the differential expression gene, can be by the protein or the multiple host animal of its partial immunity of injection differential expression gene.This class host animal can include but not limited to rabbit, mouse and the rat only mentioned.Multiple adjuvant can use to improve immune response according to host's kind, and this class adjuvant includes but not limited to human adjuvant such as the BCG (bacille Calmette-Guerin vaccine) and the CBP of Fu Shi (fully with incomplete) adjuvant, mineral rubber such as aluminium hydroxide, surfactant such as lysolecithin, Pluronic polyvalent alcohol, polyanion, peptide, oil emulsion, keyhole limpet hemocyanin, dinitrophenol and potentially useful.

Polyclonal antibody is to derive from through antigen such as target gene product or its to have the heterologous antibody molecule colony of the animal serum of antigenic functional derivatives immunity.In order to produce polyclonal antibody, aforesaid host animal can add as mentioned above the differential expression gene product of adjuvant by injection and carry out immunity.

As obtaining by any technology that is used in the continuous cell line of cultivating, producing antibody molecule at the homogeneous antibody group's of specific antigen monoclonal antibody.These technology include but not limited to Kohler and Milstein (1975, Nature 256:495-497; With U.S. Patent number 4,376,110) hybridoma technology, people B-quadroma technology (Kosbo etc., 1983, Immunology Today 4:72; Cole etc., 1983, Proc.Natl.Acad.Sci.USA 80:2026-2030) and EBV-hybridoma technology (Alan R.Liss in 1985, " Monoclonal Antibody AndCancer Therapy " 77-96 pages or leaves such as the Cole of Inc press).This antibody-like can belong to arbitrary immunoglobulins and comprise IgG, IgM, IgE, IgA, IgD and its arbitrary subclass.The hybridoma that produces mAb of the present invention can external or culturing in vivo.High titre produces and makes culturing in vivo become present preferred production methods in the mAb body.

Can use in addition research and development be used for produce the technology of " chimeric antibody " (Morrison 1984, Proc.Natl.Acad.Sci., 81:6851-6855; Neuberger etc., 1984, Nature, 312:604-608; Takeda etc., 1985, Nature, 314:452-454), this technology is by making the gene splicing in gene with the specific mouse antibodies molecule of suitable antigen source and the human antibody molecule source with suitable biologic activity.Chimeric antibody is the molecule that different piece comes from the different animals kind in a kind of molecule, has as those to derive from mouse mAb variable region or hypervariable region and the molecule with human normal immunoglobulin constant region.

Alternatively, describe with the technology (U.S. Patent number 4,946,778 that produce single-chain antibody; Bird, 1988, Science 242:423-426; Huston etc., 1988, Proc.Natl.Acad.Sci.USA85:5879-5883 and Ward etc., 1989, Nature 334:544-546) can be through adjusting to produce differential expression gene-single-chain antibody.Single-chain antibody connects the heavy chain and the light chain segments in Fv zone by the amino acid bridging, has produced single chain polypeptide and forms.

The most preferably, the technology that is used for generation " humanized antibody " can be passed through adjustment to produce the antibody at polypeptide disclosed herein, fragment, derivative and function equivalent.This class technology is in U.S. Patent number 5,932,448; 5,693,762; 5,693,761; 5,585,089; 5,530,101; 5,910,771; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,545,580; Open in 5,661,016 and 5,770,429, all these patents are intactly quoted as a reference herein.

The antibody fragment of identification specificity epi-position can produce by known technology.For example this type of fragment includes but not limited to: the F (ab ') that can produce by the gastric pepsin digestion effect of antibody molecule ₂Fragment and by F (ab ') ₂The Fab that segmental disulphide bridges reductive action produces.Alternatively can make up the Fab expression library (Huse etc., 1989, Science is 246:1275-1281) to identify having required specific mono-clonal Fab fragment fast and simply.

For especially preferred sandwich identification method easy to detect, there be numerous changing form in this identification method, and it is included that they are the present invention.

For example in typical forward identification method, unlabelled antibody is fixing and sample to be detected contacted with fixed antibody molecule on solid substrate.After time, described time durations is enough to allow to form the antigen-antibody binary complex at one section suitable incubation.Add at this moment then can import detectable signal with the second antibody of reporter molecules mark and incubation capacity time to form three former compound as antigen-antibody-through the antibody of mark.With any unreacted matters flush away and by signal observe to determine antigenic existence or by with contain the antigenic check sample of known quantity and relatively carry out quantitatively.Changing form of forward identification method comprises identification method simultaneously, sample and antibody add to fixed antibody simultaneously in the method, perhaps comprise reverse identification method, in this method through the antibody of mark at first combine with sample to be tested, incubation and add to unlabelled surface bonding antibody.These technology are that the possibility of change that the crowd knows and trickle will be conspicuous for those skilled in the art.As used herein, " sandwich identification method " is intended to comprise changing form of all basic dibit point technology.

The most frequently used reporter molecules is enzyme or contains fluorophore or the molecule of radionuclide in this type of identification method.In enzyme immunity identification method, enzyme and second antibody are puted together through glutaraldehyde or periodate method usually.Yet be easy to recognize the interconnection technique that exists wide range of types different, these technology are that those of skill in the art are well-known.Enzyme commonly used comprises horseradish peroxidase, glucose oxidase, beta galactosidase enzyme and alkaline phosphatase etc.Usually be chosen as the substrate that can detect colour-change through corresponding enzymic hydrolysis generation with the common substrate that uses of certain enzyme.Alternatively, fluorescent chemicals such as fluorescein and rhodamine can not change the binding ability of antibody with the coupling of antibody chemical.When activating by rayed through specific wavelength, absorb luminous energy through fluorescently-labeled antibody, in molecule, induce excited state, subsequently with characteristic longer wavelength emission light.Emission shows as the visually detected characteristic color of opticmicroscope.Present method is all set up and especially be preferred for to immunofluorescence and EIA technology fully in this area.Yet other reporter molecules such as radio isotope, chemoluminescence or bioluminescent molecules also can be used.How adjusting this method for technicians is conspicuous to be fit to required purposes.Therefore in yet another aspect, the present invention relates to diagnostic kit, it comprises at least a following component: the oligonucleotide that (a) is suitable for detecting the nucleic acid that comprises shown in SEQ ID No.2, SEQ ID.No.6 or SEQ ID No.10 nucleotide sequence or its part; (b) be suitable for detecting the antibody of the polypeptide that comprises shown in SEQ ID No.1, SEQ ID.No.5 or SEQ ID No.9 aminoacid sequence or its part; (c) working instructions of this test kit.

Nucleotide sequence of the present invention is for the chromosomal value that is positioned with.The specific position of this sequence-specific target on individual human chromosomal also can be hybridized with this position.Correlated series is that these sequences and gene-correlation disease are set up first important step of getting in touch according to the present invention to chromosomal genetic mapping.In case certain sequence is located on chromosomal exact position, then the physical location of this sequence on karyomit(e) can be set up with the genetic map data and get in touch.These type of data can be at for example V.McKusick, finds in the human Mendelian inheritance (can by the online acquisition of Johns Hopkins University's Welch medical library).The relation of localized gene and disease obtains identifying (the common heredity of physical property contiguous gene) by linkage analysis then in identical chromosomal region.

Difference at cDNA or genome sequence between the ill and not ill individuality also can be measured.Do not observe this sudden change in any normal individual if observed certain sudden change in some or all of diseased individuals, then this sudden change might be the paathogenic factor of this disease.

Additional aspect of the present invention relates to for any therapeutic effects as discussed above uses the pharmaceutical composition that contains pharmaceutically acceptable carrier.This class pharmaceutical composition can be made up of antibody, stand-in, agonist, antagonist or the inhibitor of ubiquitin specific protease of the present invention.This based composition can be used separately or be co-administered with at least a other reagent such as stable compound, it can be used in any aseptic, biocompatibility pharmaceutical carriers, and described aseptic, biocompatibility pharmaceutical carriers includes but not limited to salt solution, buffer saline, glucose and water.This based composition can be used separately to the patient, perhaps uses with other reagent, medicine or hormons.

The pharmaceutical composition that the present invention comprised can be used by any approach, includes but not limited in per os, in intravenous, intramuscular, IA, endarterial, the marrow, the sheath, intraventricular, in skin, subcutaneous, endoperitoneal, nose, intestines, partial, the hypogloeeis or per-rectum application process.

Except activeconstituents, these pharmaceutical compositions can comprise suitable pharmaceutically acceptable carrier, comprise to be beneficial to active compound is processed as vehicle and the auxiliary material that can make medicinal preparation.Preparation and other ins and outs of using can (MaackPublishing Co., Easton find in Pa.) at latest edition Remington ' s Pharmaceutical Sciences.

The pharmaceutical composition that is used for dosage forms for oral administration can use pharmaceutically acceptable carrier well known in the art to be suitable for the dosage particles of dosage forms for oral administration.It is that tablet, pill, coated tablet, capsule, liquid agent, gelifying agent, syrup, paste, suspension etc. are so that patient's picked-up that this class carrier can make drug combination preparation.

The pharmaceutical preparation of dosage forms for oral administration can obtain by the combination of active compound and solid excipient, randomly grind the mixture that obtains and as required after adding proper supplementary material with compound particles processing to obtain tablet or dragee cores.Suitable vehicle is carbohydrate or protein weighting material such as sugar, comprises lactose, sucrose, mannitol, Sorbitol Powder; Starch from cereal, wheat, rice, potato or other plant; Mierocrystalline cellulose such as methylcellulose gum, Vltra tears or Xylo-Mucine; Natural gum comprises Sudan Gum-arabic and Tragacanth; With protein such as gelatin and collagen.As required, can add dispersion agent and solubilizing agent such as crosslinked polyvinylpyrrolidone, agar, alginic acid or its salt such as sodium alginate.

Dragee cores can be used for combining with suitable dressing such as spissated sugar soln, and described dressing can also comprise Sudan Gum-arabic, talcum powder, polyvinylpyrrolidone, carbopol gel, polyoxyethylene glycol and/or titanium dioxide, lacquer solution and appropriate organic solvent or solvent mixture.Can in tablet or coated tablet dressing, add dyestuff and pigment so that product is identified or the qualitative characteristics of description active compound is a dosage.

But the pharmaceutical preparation of dosage forms for oral administration comprises the capsule of the sucking fit of being made by gelatin, and the soft seal capsule of being made by gelatin and dressing such as glycerine or Sorbitol Powder.The capsule of sucking fit can comprise with weighting material or tackiness agent such as lactose or starch, lubricant such as talcum powder or Magnesium Stearate and randomly with stablizer blended activeconstituents.In soft capsule, can or be suspended in the suitable liquid that has or do not have stablizer the active compound dissolving as in fatty oil, liquid or the liquid polyethylene glycol.

Be suitable for the pharmaceutical preparation of parenteral administration can be in the aqueous solution preparation, preferably preparation in the compatible damping fluid of physiology such as Hanks liquid, Ringer's solution or physiological buffer salt solution.Moisture injection suspension can comprise the material that increases suspension viscosity, as Xylo-Mucine, Sorbitol Powder or dextran.The suspension of active compound can be prepared as suitable oily injection suspension in addition.Suitable lipophilic solvent or carrier comprise fatty oil such as sesame oil or synthetic fatty acid ester such as ethyl oleate or triglyceride level or liposome.The amino polymer of the polycation of non-fat also can be used for sending.Randomly, suspension also can comprise the reagent of suitable stabilizers or enhancing compound dissolution to allow the highly spissated solution of preparation.

Use for part or nose, in preparation, used the permeate agent that is suitable for permeating particular barrier.This class permeate agent is generally known in the art.

Pharmaceutical composition of the present invention can be made according to mode known in the art, for example by routine mixing, dissolving, granulation, manufacturing drageeing, grinding, emulsification, incapsulate, seal or freeze drying process.

Pharmaceutical composition can be used as that salt provides and can form salt with multiple acid, and described acid includes but not limited to hydrochloric acid, sulfuric acid, acetate, lactic acid, tartrate, oxysuccinic acid, succsinic acid etc.Salt is more more solvable in water solvent or other protic solvent than corresponding free alkali form.In other cases, preferred goods can be lyophilized powders, this lyophilized powder can comprise any or whole following materials: 1-50mM Histidine, 0.1%-2% sucrose and 2-7% mannitol, 4.5 to 5.5 pH scope, these materials before use with combinations of buffers.

Pharmaceutical composition can place suitable containers and mark to be used for the treatment of indicated disease after preparation.For using, mark comprises amount of application, frequency of administration and application process.

Be applicable to that pharmaceutical composition of the present invention comprises such composition, contain effective amount of actives in the described composition to realize its intended purposes.Significant quantity be defined as those skilled in the art's capabilities.

For arbitrary compound, the treatment significant quantity can at first be assessed in the cell cultures identification method, as in oncocyte, or assesses in mouse, rabbit, dog or pig usually at animal model.Animal model can also be used to determine suitable concn scope and the approach used.This type of information can be used for determining to be applied to people's useful dosage and approach then.

The treatment significant quantity means the amount of activeconstituents or its fragment, antibody, agonist, antagonist or the inhibitor of ubiquitin specific protease, and described amount is improved symptom or disease.Result of treatment and toxicity can be measured in cell culture or laboratory animal by the method for pharmacy of standard, as ED50 (the effective dosage of treatment in 50% colony) and LD50 (to the lethal dosage of 50% colony).Dosage ratio between toxic effect and the result of treatment is therapeutic index and can be expressed as ratio LD50/ED50.Preferably show big treatment exponential pharmaceutical composition.The data that obtain from cell cultures identification method and zooscopy can be used for forming the dosage range of human.The dosage that contains in this based composition preferably is in and comprises ED50 and have few or do not have in the toxic circulation composition scope.According to used formulation, patient's susceptibility and route of administration, dosage can change in this scope.

Precise dosage will be determined by the practitioner, consider and the relevant factor of experimenter that needs treatment.Dosage and using through adjusting with bioactive molecule that enough levels are provided or to keep required effect.The factor that can consider comprises the seriousness of morbid state, experimenter's general health, subject age, body weight and sex, diet, the time of using and frequency, drug combination, reaction sensibility and the tolerance/reaction to treating.The depot drug product composition can or be used once according to half life and per 3 to 4 days of clearance rate, the per week of particular formulations per fortnight.

Usually dosage can be according to route of administration in the change between about 1 gram to the total dose as many as of 0.1 to 100,000 microgram.The guidance of given dose and delivering method is provided and to be generally the practitioner of this area obtainable in the literature.Those skilled in the art will adopt the preparation that is different from protein or its inhibitor to Nucleotide.Similarly, sending polynucleotide or polypeptide is specific for specific cells, disease, position etc.The pharmaceutical preparation that is applicable to the protein dosage forms for oral administration is at for example United States Patent (USP) 5,008,114; 5,505,962; 5,641,515; 5,681,811; 5,700,486; 5,766,633; 5,792,451; 5,853,748; 5,972,387; Describe in 5,976,569 and 6,051,561.

Following embodiment and explanation of tables the present invention, in any case but do not limit the scope of the invention.

Table:

Table 1 (a)-USP_N01: new splice variant-aminoacid sequence

Locus USP_N01 3370aa PRT

Accession number BAA25496

GeneSeq AAU82706

People from source

Insert the splicing form that characterizes with 4:

Pos?1-Pos?11

Pos?267-Pos?300

Pos?361-Pos?384

Pos?1243-Pos?1437

1 MRRKNSYYVW?QKIFQIQFPL?YTAYKHNTHP?TIEDISTQES?NILGAFCDMN?DVEVPLHLLR

61 YVCLFCGKNG?LSLMKDCFEY?GTPETLPFLI?AHAFITVVSN?IRIWLHIPAV?MQHIIPFRTY

121 VIRYLCKLSD?QELRQSAARN?MADLMWSTVK?EPLDTTLCFD?KESLDLAFKY?FMSPTLTMRL

181 AGLSQITNQL?HTFNDVCNNE?SLVSDTETSI?AKELADWLIS?NNVVEHIFGP?NLHIEIIKQC

241 QVILNFLAAE?GRLSTQHIDC?IWAAAQRKKS?LEEQLHHLGH?LQLVLKAVII?AIHIKVEVVT

301 EPSVHTEQTL?YLASMLIKAL?WNNALAAKAQ?LSKQSSFASL?LNTNIPIGNK?KEEEELRRTA

361 LKWMSNLLIE?PNMCNNDFQT?QKESMQGSSD?ETANSGEDGS?SGPGSSSGHS?DGSSNEVNSS

421 HASQSAGSPG?SEVQSEDIAD?IEALKEEDED?DDHGHNPPKS?SCGTDLRNRK?LESQAGICLG

481 DSQGTSERNG?TSSGTGKDLV?FNTESLPSVD?NRMRMLDACS?HSEDPEHDIS?GEMNATHIAQ

541 GSQESCITRT?GDFLGETIGN?ELFNCRQFIG?PQHHHHHHHH?HHHHDGHMVD?DMLSADDVSC

601 SSSQVSAKSE?KNMADFDGEE?SGCEEELVQI?NSHAELTSHL?QQHLPNLASI?YHEHLSQGPV

661 VHKHQFNSNA?VTDINLDNVC?KKGNTLLWDI?VQDEDAVNLS?EGLINEAEKL?LCSLVCWFTD

721 RQIRMRFIEG?CLENLGNNRS?VVISLRLLPK?LFGTFQQFGS?SYDTHWITMW?AEKELNMMKL

781 FFDNLVYYIQ?TVREGRQKHA?LYSHSAEVQV?RLQFLTCVFS?TLGSPDHFRL?SLEQVDILWH

841 CLVEDSECYD?DALHWFLNQV?RSKDQHAMGM?ETYKHLFLEK?MPQLKPETIS?MTGLNLFQHL

901 CNLARLATSA?YDGCSNSELC?GMDQFWGIAL?RAQSGDVSRA?AIQYINSYYI?NGKTGLEKEQ

961 EFISKCMESL?MIASSSLEQE?SHSSLMVIER?GLLMLKTHLE?AFRRRFAYHL?RQWQIEGTGI

1021?SSHLKALSDK?QSLPLRVVCQ?PAGLPDKMTI?EMYPSDQVAD?LRAEVTHWYE?NLQKEQINQQ

1081?AQLQEFGQSN?RKGEFPGGLM?GPVRMISSGH?ELTTDYDEKA?LHELGFKDMQ?MVFVSLGAPR

1141?RERKGEGVQL?PASCLPPPQK?DNIPMLLLLQ?EPHLTTLFDL?LEMLASFKPP?SGKVAVDDSE

1201?SLRCEELHLH?AENLSRRVWE?LLMLLPTCPN?MLMAFQNISD?EQSNDGFNWK?ELLKIKSAHK

1261?LLYALEIIEA?LGKPNRRIRR?ESTGSYSDLY?PDSDDSSEDQ?VENSKNSWSC?KFVAAGGLQQ

1321?LLEIFNSGIL?EPKEQESWTV?WQLDCLACLL?KLICQFAVDP?SDLDLAYHDV?FAWSGIAESH

1381?RKRTWPGKSR?KAAGDHAKGL?HIPRLTEVFL?VLVQGTSLIQ?RLMSVAYTYD?NLAPRVLKAQ

1441?SDHRSRHEVS?HYSMWLLVSW?AHCCSLVKSS?LADSDHLQDW?LKKLTLLIPE?TAVRHESCSG

1501?LYKLSLSGLD?GGDSINRSFL?LLAASTLLKF?LPDAQALKPI?RIDDYEEEPI?LKPGCKEYFW

1561?LLCKLVDNIH?IKDASQTTLL?DLDALARHLA?DCIRSREILD?HQDGNVEDDG?LTGLLRLATS

1621?VVKHKPPFKF?SREGQEFLRD?IFNLLFLLPS?LKDRQQPKCK?SHSSRAAAYD?LLVEMVKGSV

1681?ENYRLIHNWV?MAQHMQSHAP?YKWDYWPHED?VRAECRFVGL?TNLGATCYLA?STIQQLYMIP

1741?EARQAVFTAK?YSEDMKHKTT?LLELQKMFTY?LMESECKAYN?PRPFCKTYTM?DKQPLNTGEQ

1801?KDMTEFFTDL?ITKIEEMSPE?LKNTVKSLFG?GVITNNVVSL?DCEHVSQTAE?EFYTVRCQVA

1861?DMKNIYESLD?EVTIKDTLEG?DNMYTCSHCG?KKVRAEKRAC?FKKLPRILSF?NTMRYTFNMV

1921?TMMKEKVNTH?FSFPLRLDMT?PYTEDFLMGK?SERKEGFKEV?SDHSKDSESY?EYDLIGVTVH

1981?TGTADGGHYY?SFIRDIVNPH?AYKNNKWYLF?NDAEVKPFDS?AQLASECFGG?EMTTKTYDSV

2041?TDKFMDFSFE?KTHSAYMLFY?KRMEPEEENG?REYKFDVSSE?LLEWIWHDNM?QFLQDKNIFE

2101?HTYFGFMWQL?CSCIPSTLPD?PKAVSLMTAK?LSTSFVLETF?IHSKEKPTML?QWIELLTKQF

2161?NNSQAACEWF?LDRMADDDWW?PMQILIKCPN?QIVRQMFQRL?CIHVIQRLRP?VHAHLYLQPG

2221?MEDGSDDMDT?SVEDIGGRSC?VTRFVRTLLL?IMEHGVKPHS?KHLTEYFAFL?YEFAKMGEEE

2281?SQFLLSLQAI?STMVHFYMGT?KGPENPQVEV?LSEEEGEEEE?EEEDILSLAE?EKYRPAALEK

2341?MIALVALLVE?QSRSERHLTL?SQTDMAALTG?GKGFPFLFQH?IRDGINIRQT?CNLIFSLCRY

2401?NNRLAEHIVS?MLFTSIAKLT?PEAANPFFKL?LTMLMEFAGG?PPGMPPFASY?ILQRIWEVIE

2461?YNPSQCLDWL?AVQTPRNKLA?HSWVLQNMEN?WVERFLLAHN?YPRVRTSAAY?LLVSLIPSNS

2521?FRQMFRSTRS?LHIPTRDLPL?SPDTTVVLHQ?VYNVLLGLLS?RAKLYVDAAV?HGTTKLVPYF

2581?SFMTYCLISK?TEKLMFSTYF?MDLWNLFQPK?LSEPAIATNH?NKQALLSFWY?NVCADCPENI

2641?RLIVQNPVVT?KNIAFNYILA?DHDDQDVVLF?NRGMLPAYYG?ILRLCCEQSP?AFTRQLASHQ

2701?NIQWAFKNLT?PHASQYPGAV?EELFNLMQLF?IAQRPDMREE?ELEDIKQFKK?TTISCYLRCL

2761?DGRSCWTTLI?SAFRILLESD?EDRLLVVFNR?GLILMTESFN?TLHMMYHEAT?ACHVTGDLVE

2821?LLSIFLSVLK?STRPYLQRKD?VKQALIQWQE?RIEFAHKLLT?LLNSYSPPEL?RNACIDVLKE

2881?LVLLSPHDFL?HTLVPFLQHN?HCTYHHSNIP?MSLGPYFPCR?ENIKLIGGKS?NIRPPRPELN

2941?MCLLPTMVET?SKGKDDVYDR?MLLDYFFSYH?QFIHLLCRVA?INCEKFTETL?VKLSVLVAYE

3001?GLPLHLALFP?KLWTELCQTQ?SAMSKNCIKL?LCEDPVFAEY?IKCILMDERT?FLNNNIVYTF

3061?MTHFLLKVQS?QVFSEANCAN?LISTLITNLI?SQYQNLQSDF?SNRVEISKAS?ASLNGDLRAL

3121?ALLLSVHTPK?QLNPALIPTL?QELLSKCRTC?LQQRNSLQEQ?EAKERKTKDD?EGATPIKRRR

3181?VSSDEEHTVD?SCISDMKTET?REVLTPTSTS?DNETRDSSII?DPGTEQDLPS?PENSSVKEYR

3241?MEVPSSFSED?MSNIRSQHAE?EQSNNGRYDD?CKEFKDLHCS?KDSTLAEEES?EFPSTSISAV

3301?LSDLADLRSC?DGQALPSQDP?EVALSLSCGH?SRGLFSHMQQ?HDILDTLCRT?IESTIHVVTR

3361?ISGKGNQAAS

Table 1 (b) :-USP_N01: new splice variant-nucleotide sequence

1 atgagaagga?aaaactctta?ctatgtgtgg?caaaaaattt?ttcaaattca?gtttccctta

61 tatactgctt?acaagcataa?tactcaccct?actattgagg?atatatcaac?tcaagaaagt

121 aacatattag?gggcattctg?tgatatgaat?gatgtagaag?taccattgca?tttgcttcgt

181 tatgtatgtt?tgttttgtgg?gaaaaatggc?ctttctctca?tgaaggattg?ctttgaatat

241 ggaactcctg?aaactttgcc?atttcttata?gcacatgcgt?ttattacagt?tgtgtctaat

301 attagaatat?ggctacatat?tcccgctgtc?atgcagcaca?ttataccttt?taggacctat

361 gttattaggt?atttatgcaa?gctctcggat?caggagttac?gacagagtgc?agctcgtaac

421 atggctgact?taatgtggag?cacagtcaaa?gaaccattgg?atacaacatt?atgctttgat

481 aaagaaagcc?tagatcttgc?atttaagtac?tttatgtcac?ctactttgac?tatgaggttg

541 gctggattga?gtcagataac?aaatcaactc?cataccttca?atgatgtgtg?caataatgaa

601 tcattagtat?cggacacaga?aacgtccatt?gcaaaagaac?ttgcagactg?gcttattagc

661 aacaatgtgg?tggagcatat?atttggacca?aatttacata?ttgagattat?caaacagtgc

721 caagtgattt?tgaatttttt?ggcagcagaa?gggcgactga?gtactcaaca?tattgactgt

781 atttgggctg?cagcacagag?gaagaagagc?ttagaagaac?agctccatca?ccttggtcac

841 ctgcagctag?tcctcaaagc?agtgataata?gcgatacaca?tcaaagtgga?ggtagtgaca

901 ttgaaatgga?tgagcaactt?attaatagaa?ccaaacatgt?gcaacaacga?ctttcagaca

961 cagaaggaat?ccatgcaggg?aagttctgac?gaaactgcca?acagtggtga?agatggaagc

1021?agtggtcctg?gtagcagtag?tgggcatagt?gatggatcta?gcaatgaggt?taattctagc

1081?cacgcaagcc?agtcagctgg?gagccctggc?agtgaggtac?agtcagaaga?cattgcagat

1141?attgaagccc?tcaaagagga?agatgaagac?gatgatcatg?gtcataatcc?tcccaaaagc

1201?agttgtggta?cagatcttcg?gaatagaaag?ttagagagtc?aagcaggcat?ttgcctgggg

1261?gactcccaag?gcacgtcaga?aagaaatggg?acaagcagcg?gaacaggaaa?ggacctggtt

1321?tttaacactg?aatcattgcc?atcagtagat?aatcgaatgc?gaatgctgga?tgcttgttca

1381?cactctgaag?acccagaaca?tgatatttca?ggggaaatga?atgctactca?tatagcacaa

1441?gggtctcagg?agtcttgtat?cacacgaact?ggggacttcc?ttggggagac?tattgggaat

1501?gaattattta?attgtcgaca?atttattggt?ccacagcatc?accaccacca?ccaccaccat

1561?caccaccacc?acgatgggca?tatggttgat?gatatgctaa?gtgcagatga?tgtcagttgt

1621?agtagctccc?aggttagtgc?aaaatcagaa?aaaaatatgg?ctgattttga?tggtgaagaa

1681?tctggatgtg?aagaggagct?agttcagatt?aattcacatg?cggaactgac?atctcacctc

1741?caacaacatc?ttcccaattt?agcttccatt?taccatgaac?atcttagtca?aggacctgta

1801?gttcataaac?atcaattcaa?cagtaatgct?gttacagaca?ttaatttgga?taatgtttgc

1861?aagaaaggaa?atactttgtt?gtgggatata?gtccaagatg?aagatgcagt?taatctttct

1921?gaaggattaa?taaatgaagc?agagaaactt?ctttgttcgt?tagtatgttg?gtttacagat

1981?agacaaattc?gaatgagatt?cattgaaggt?tgccttgaaa?acttgggaaa?caacagatca

2041?gtagtaattt?cacttcgtct?tcttccaaaa?ctatttggta?cttttcagca?gtttgggagc

2101?agttacgata?cacactggat?aacaatgtgg?gcagaaaaag?aactgaacat?gatgaagctt

2161?ttctttgata?atttggtata?ctacattcaa?actgtgagag?aaggaagaca?aaaacatgca

2221?ctgtacagcc?atagtgctga?agttcaagtt?cgtcttcaat?tcttgacttg?tgtattttca

2281?actctgggat?cacctgatca?tttcaggtta?agtttagagc?aagttgacat?cttatggcat

2341?tgtttagtag?aagattctga?atgttatgat?gatgcactcc?attggttttt?aaatcaagtt

2401?cgaagtaaag?atcaacatgc?tatgggtatg?gaaacctaca?aacatctttt?cctggagaag

2461?atgccccagc?taaaacctga?aacaattagc?atgactggct?taaacctgtt?tcagcatctc

2521?tgtaacttgg?ctcgattggc?taccagtgcc?tatgatggtt?gttcaaattc?tgagctgtgt

2581?ggtatggacc?aattttgggg?cattgcttta?agagcacaat?ctggtgatgt?cagtcgagca

2641?gctatccagt?atattaactc?ctattatatt?aatggtaaaa?caggtttgga?gaaggagcaa

2701?gaatttatta?gtaagtgcat?ggagagtctt?atgatagctt?ctagcagtct?tgaacaggaa

2761?tcacactcaa?gtctcatggt?tatagaaaga?ggactcctta?tgctgaagac?acatctggaa

2821?gcgtttagga?gaaggtttgc?atatcatctg?agacagtggc?aaattgaagg?cactggtatt

2881?agtagtcatt?tgaaagcact?gagtgacaaa?cagtctctgc?cgctaagggt?tgtatgccag

2941?ccagctggac?ttcctgacaa?gatgactatt?gaaatgtatc?ctagtgacca?ggtagcagat

3001?cttagggctg?aagtaactca?ttggtatgaa?aatttacaga?aagaacaaat?aaatcaacaa

3061?gctcagcttc?aggagtttgg?tcaaagcaac?cgaaaaggag?agtttcctgg?aggcctcatg

3121?ggacctgtca?ggatgatttc?atctggacac?gagttaacaa?cagattatga?tgaaaaagca

3181?cttcatgagc?ttggttttaa?ggatatgcag?atggtatttg?tatctttggg?tgcaccaagg

3241?agagagcgga?aaggggaagg?tgttcagctg?ccagcatctt?gcctcccacc?ccctcagaag

3301?gacaacattc?caatgctttt?gcttttacaa?gagcctcatt?taactactct?ttttgattta

3361?ttagagatgc?ttgcatcatt?taaaccaccc?tcaggaaaag?tggcagtgga?tgatagtgag

3421?agcttacgat?gtgaagaact?tcatcttcat?gcagaaaatc?tgtctaggcg?ggtctgggag

3481?ctactgatgc?ttcttcctac?atgtcctaat?atgttgatgg?cattccagaa?tatctcagat

3541?gagcagagta?atgatggatt?taattggaaa?gaacttctca?aaattaagag?cgcccacaag

3601?ctattgtatg?ctctggaaat?tattgaagca?ctgggaaaac?ctaatagaag?aataaggagg

3661?gagtctacgg?gaagttacag?tgatctttat?ccagattcag?atgattcaag?tgaggatcaa

3721?gtggaaaata?gtaaaaattc?ctggagttgc?aagtttgttg?ctgctggagg?gcttcaacag

3781?ttattagaaa?tttttaattc?tggaattcta?gagcctaaag?agcaggaatc?atggactgtg

3841?tggcagctag?actgtcttgc?ttgcttgctg?aagttaatat?gccagtttgc?agtagatcca

3901?tccgatttgg?atttagctta?tcatgatgtc?tttgcctggt?ctggtatagc?ggaaagccat

3961?aggaaaagaa?cctggcctgg?caaatcaagg?aaggctgctg?gtgatcatgc?taagggtctt

4021?catataccac?gattaacaga?ggtatttctt?gttcttgtcc?aaggaaccag?tttgattcag

4081?cgacttatgt?ctgttgctta?tacgtatgat?aatctggctc?ctagagtttt?aaaagctcag

4141?tctgatcaca?ggtctagaca?tgaagtttca?cattattcaa?tgtggctctt?ggtgagttgg

4201?gctcattgct?gttctttagt?gaaatctagc?cttgctgata?gcgatcattt?acaagattgg

4261?ctaaagaaat?tgactctcct?tattcctgag?actgcagttc?gtcatgaatc?atgcagtggt

4321?ctctataagt?tatccctgtc?agggctggat?ggaggagact?caatcaatcg?ttcttttctg

4381?ctattggctg?cctcaacatt?attgaaattt?cttcctgatg?ctcaagcact?caaacctatt

4441?aggatagatg?attatgagga?agaaccaata?ttaaaaccag?gatgtaaaga?gtatttttgg

4501?ttgttatgca?aattagttga?caacatacat?ataaaggacg?ctagtcagac?aacgctcctc

4561?gacttagatg?ccttggcaag?acatttggct?gactgtattc?gaagtaggga?gatccttgat

4621?catcaggatg?gtaatgtaga?agatgatggg?cttacaggac?tcctaaggct?tgcaacaagt

4681?gttgttaaac?acaaaccacc?ctttaaattt?tcaagggaag?gacaggaatt?tttgagagat

4741?atcttcaatc?tcctgttttt?gttgccaagt?ctaaaggacc?gacaacagcc?aaagtgcaaa

4801?tcacattctt?caagagctgc?cgcttacgat?ttgttagtag?agatggtaaa?ggggtctgtt

4861?gagaactaca?ggctaataca?caactgggtt?atggcacaac?acatgcagtc?ccatgcacct

4921?tataaatggg?attactggcc?tcatgaagat?gtccgtgctg?aatgtagatt?tgttggcctt

4981?actaaccttg?gagctacttg?ttacttagct?tctactattc?agcaacttta?tatgatacct

5041?gaggcaagac?aggctgtctt?cactgccaag?tattcagagg?atatgaagca?caagaccact

5101?cttctggagc?ttcagaaaat?gtttacatat?ttaatggaga?gtgaatgcaa?agcatataat

5161?cctagacctt?tctgtaaaac?atacaccatg?gataagcagc?ctctgaatac?tggggaacag

5221?aaagatatga?cagagttttt?tactgatcta?attaccaaaa?tcgaagaaat?gtctcccgaa

5281?ctgaaaaata?ccgtcaaaag?tttatttgga?ggtgtaatta?caaacaatgt?tgtatccttg

5341?gattgtgaac?atgttagtca?aactgctgaa?gagttttata?ctgtgaggtg?ccaagtggct

5401?gatatgaaga?acatttatga?atctcttgat?gaagttacta?taaaagacac?tttggaaggt

5461?gataacatgt?atacttgttc?tcattgtggg?aagaaagtac?gagctgaaaa?aagggcatgt

5521?tttaagaaat?tgcctcgcat?tttgagtttc?aatactatga?gatacacatt?taatatggtc

5581?acgatgatga?aagagaaagt?gaatacacac?ttttccttcc?cattacgttt?ggacatgacg

5641?ccctatacag?aagattttct?tatgggaaag?agtgagagga?aagaaggttt?taaagaagtc

5701?agtgatcatt?caaaagactc?agagagctat?gaatatgact?tgataggagt?gactgttcac

5761?acaggaacgg?cagatggtgg?acactattat?agctttatca?gagatatagt?aaatccccat

5821?gcttataaaa?acaataaatg?gtatcttttt?aatgatgctg?aggtaaaacc?ttttgattct

5881?gctcaacttg?catctgaatg?ttttggtgga?gagatgacga?ccaagaccta?tgattctgtt

5941?acagataaat?ttatggactt?ctcttttgaa?aagacacaca?gtgcatatat?gctgttttac

6001?aaacgcatgg?aaccagagga?agaaaatggc?agagaataca?aatttgatgt?ttcgtcagag

6061?ttactagagt?ggatttggca?tgataacatg?cagtttcttc?aagacaaaaa?catttttgaa

6121?catacatatt?ttggatttat?gtggcaattg?tgtagttgta?ttcccagtac?attaccagat

6181?cctaaagctg?tgtccttaat?gacagcaaag?ttaagcactt?cctttgtcct?agagacattt

6241?attcattcta?aagaaaagcc?cacgatgctt?cagtggattg?aactgttgac?gaaacagttt

6301?aataatagtc?aggcagcttg?tgagtggttt?ttagatcgta?tggctgatga?cgactggtgg

6361?ccaatgcaga?tactaattaa?gtgccctaat?caaattgtga?gacagatgtt?tcagcgtttg

6421?tgtatccatg?tgattcagag?gctgagacct?gtgcatgctc?atctctattt?gcagccagga

6481?atggaagatg?ggtcagatga?tatggatacc?tcagtagaag?atattggtgg?tcgttcatgt

6541?gtcactcgct?ttgtgagaac?cctgttatta?attatggaac?atggtgtaaa?acctcacagt

6601?aaacatctta?cagagtattt?tgccttcctt?tacgaatttg?caaaaatggg?tgaagaagag

6661?agccaatttt?tgctttcatt?gcaagctata?tctacaatgg?tacattttta?catgggaaca

6721?aaaggacctg?aaaatcctca?agttgaagtg?ttatcagagg?aagaagggga?agaagaagag

6781?gaggaagaag?atatcctctc?tctggcagaa?gaaaaataca?ggccagctgc?ccttgaaaag

6841?atgatagctt?tagttgctct?tttggttgaa?cagtctcgat?cagaaaggca?tttgacatta

6901?tcacagactg?acatggcagc?attaacagga?ggaaagggat?ttcccttctt?gtttcaacat

6961?attcgtgatg?gcatcaatat?aagacaaact?tgtaatctga?ttttcagcct?gtgtcgatac

7021?aataatcgac?ttgcagaaca?tattgtatct?atgcttttca?catcaatagc?aaagttgact

7081?cctgaggcag?ccaatccttt?ctttaagttg?ttgactatgc?taatggagtt?tgctggtgga

7141?cctccaggaa?tgcctccctt?tgcatcttat?attctgcaga?ggatatggga?ggtgattgaa

7201?tacaatcctt?ctcagtgtct?agattggttg?gcagtgcaga?caccccgaaa?taaactggca

7261?cacagctggg?tcttacagaa?tatggaaaac?tgggtcgagc?ggtttctttt?ggctcacaat

7321?tatcctagag?tgaggacttc?tgcagcttat?cttctggtgt?cccttatacc?aagcaattca

7381?ttccgtcaga?tgttccggtc?aacaaggtct?ttgcacatcc?caacccgtga?ccttccactc

7441?agtccagaca?caacagtagt?cctacatcag?gtctacaacg?tgctccttgg?tttgctctca

7501?agagccaaac?tttatgttga?tgctgctgtt?catggcacta?caaagctagt?gccctatttt

7561?agctttatga?cttactgttt?aatttccaaa?actgagaagc?tgatgttttc?cacatatttc

7621?atggatttgt?ggaacctttt?ccagcctaaa?ctttctgagc?cagcaatagc?tacaaatcac

7681?aataaacagg?ctttgctttc?attttggtac?aatgtctgtg?ctgactgtcc?agagaatatc

7741?cgccttattg?ttcagaaccc?agtggtaacc?aagaacattg?ccttcaatta?catccttgct

7801?gaccatgatg?atcaggatgt?ggtgcttttt?aaccgtggga?tgctgccagc?gtactatggc

7861?attctgaggc?tctgctgtga?gcagtctcct?gcattcacac?gacaactggc?ttctcaccag

7921?aacatccagt?gggcctttaa?gaatcttaca?ccacatgcca?gccaataccc?tggagcagta

7981?gaagaactgt?ttaacctgat?gcagctgttt?atagctcaga?ggccagatat?gagagaagaa

8041?gaattagaag?atattaaaca?gttcaagaaa?acaaccataa?gttgttactt?acgttgctta

8101?gatggccgct?cctgctggac?tactttaata?agtgccttca?gaatactatt?agaatctgat

8161?gaagacagac?ttcttgttgt?atttaatcga?ggattgattc?taatgacaga?gtctttcaac

8221?actttgcaca?tgatgtatca?cgaagctaca?gcttgccatg?tgactggaga?tttagtagaa

8281?cttctgtcaa?tatttctttc?ggttttgaag?tctacacgcc?cttatcttca?gagaaaagat

8341?gtgaaacaag?cattaatcca?gtggcaggag?cgaattgaat?ttgcccataa?actgttaact

8401?cttcttaatt?cctatagtcc?tccagaactt?agaaatgcct?gtatagatgt?cctcaaggaa

8461?cttgtacttt?tgagtcccca?tgattttctt?catactctgg?ttccctttct?acaacacaac

8521?cattgtactt?accatcacag?taatatacca?atgtctcttg?gaccttattt?cccttgtcga

8581?gaaaatatca?agctaatagg?agggaaaagc?aatattcggc?ctccgcgccc?tgaactcaat

8641?atgtgcctct?tgcccacaat?ggtggaaacc?agtaagggca?aagatgacgt?ttatgatcgt

8701?atgctgctag?actacttctt?ttcttatcat?cagttcatcc?atctattatg?ccgagttgca

8761?atcaactgtg?aaaaatttac?tgaaacatta?gttaagctga?gtgtcctagt?tgcctatgaa

8821?ggtttgccac?ttcatcttgc?actgttcccc?aaactttgga?ctgagctatg?ccagactcag

8881?tctgctatgt?caaaaaactg?catcaagctt?ttgtgtgaag?atcctgtttt?cgcagaatat

8941?attaaatgta?tcctaatgga?tgaaagaact?tttttaaaca?acaacattgt?ctacacgttc

9001?atgacacatt?tccttctaaa?ggttcaaagt?caagtgtttt?ctgaagcaaa?ctgtgccaat

9061?ttgatcagca?ctcttattac?aaacttgata?agccagtatc?agaacctaca?gtctgatttc

9121?tccaaccgag?ttgaaatttc?caaagcaagt?gcttctttaa?atggggacct?gagggcactc

9181?gctttgctcc?tgtcagtaca?cactcccaaa?cagttaaacc?cagctctaat?tccaactctg

9241?caagagcttt?taagcaaatg?caggacttgt?ctgcaacaga?gaaactcact?ccaagagcaa

9301?gaagccaaag?aaagaaaaac?taaagatgat?gaaggagcaa?ctcccattaa?aaggcggcgt

9361?gttagcagtg?atgaggagca?cactgtagac?agctgcatca?gtgacatgaa?aacagaaacc

9421?agggaggtcc?tgaccccaac?gagcacttct?gacaatgaga?ccagagactc?ctcaattatt

9481?gatccaggaa?ctgagcaaga?tcttccttcc?cctgaaaata?gttctgttaa?agaataccga

9541?atggaagttc?catcttcgtt?ttcagaagac?atgtcaaata?tcaggtcaca?gcatgcagaa

9601?gaacagtcca?acaatggtag?atatgacgat?tgtaaagaat?ttaaagacct?ccactgttcc

9661?aaggattcta?ccctagctga?ggaagaatct?gagttccctt?ctacttctat?ctctgcagtt

9721?ctgtctgact?tagctgactt?gagaagctgt?gatggccaag?ctttgccctc?ccaggaccct

9781?gaggttgctt?tatctctcag?ttgtggccat?tccagaggac?tctttagtca?tatgcagcaa

9841?catgacattt?tagataccct?gtgtaggacc?attgaatcta?caatccatgt?cgtcacaagg

9901?atatctggca?aaggaaacca?agctgcttct?tga

Table 1 (c)-USP_N01 reference sequences (Derwent AAU82706)

Aminoacid sequence:

1 mcencadlve?vlneisdveg?gdglqlrkeh?tlkiftyins?wtqrqclccf?keykhleifn

61 qvvcalinlv?iaqvqvlrdq?lckhcttini?dstwqdesnq?aeeplnidre?cnegsterqk

121 siekksnstr?icnlteeess?kssdpfslws?tdekeklllc?vakifqiqfp?lytaykhnth

181 ptiedistqe?snilgafcdm?ndvevplhll?ryvclfcgkn?glslmkdcfe?ygtpetlpfl

241 iahafitvvs?niriwlhipa?vmqhiipfrt?yvirylckls?dqelrqsaar?nmadlmwstv

301 kepldttlcf?dkesldlafk?yfmsptltmr?laglsqitnq?lhtfndvcnn?eslvsdtets

361 iakeladwli?snnvvehifg?pnlhieiikq?cqvilnflaa?egrlstqhid?ciwaaaqlkh

421 csryihdlfp?sliknldpvp?lrhllnlvsa?lepsvhteqt?lylasmlika?lwnnalaaka

481 qlskqssfas?llntnipign?kkeeeelrrt?apspwspaas?pqssdnsdth?qsggsdiemd

541 eqlinrtkhv?qqrlsdtees?mqgssdetan?sgedgssgpg?sssghsdgss?nevnsshasq

601 sagspgsevq?sediadieal?keededddhg?hnppksscgt?dlrnrklesq?agiclgdsqg

661 tserngtssg?tgkdlvfnte?slpsvdnrmr?mldacshsed?pehdisgemn?athiaqgsqe

721 scitrtgdfl?getignelfn?crqfigpqhh?hhhhhhhhhh?dghmvddmls?addvscsssq

781 vsakseknma?dfdgeesgce?eelvqinsha?eltshlqqhl?pnlasiyheh?lsqgpvvhkh

841 qfnsnavtdi?nldnvckkgn?tllwdivqde?davnlsegli?neaekllcsl?vcwftdrqir

901 mrfiegclen?lgnnrsvvis?lrllpklfgt?fqqfgssydt?hwitmwaeke?lnmmklffdn

961 lvyyiqtvre?grqkhalysh?saevqvrlqf?ltcvfstlgs?pdhfrlsleq?vdilwhclve

1021?dsecyddalh?wflnqvrskd?qhamgmetyk?hlflekmpql?kpetismtgl?nlfqhlcnla

1081?rlatsaydgc?snselcgmdq?fwgialraqs?gdvsraaiqy?insyyingkt?glekeqefis

1141?kcmeslmias?ssleqeshss?lmviergllm?lkthleafrr?rfayhlrqwq?iegtgisshl

1201?kalsdkqslp?lrvvcqpagl?pdkmtiemyp?sdqvadlrae?vthwyenlqk?eqinqqaqlq

1261?efgqsnrkge?fPgglmgpvr?missgheltt?dydekalhel?gfkdmqmvfv?slgaprrerk

1321?gegvqlpasc?lpppqkdnip?mllllqephl?ttlfdlleml?asfkppsgkv?avddseslrc

1381?eelhlhaenl?srrvwellml?lptcpnmlma?fqnisdeqsf?kaqsdhrsrh?evshysmwll

1441?vswahccslv?kssladsdhl?qdwlkkltll?ipetavrhes?csglyklsls?gldggdsinr

1501?sflllaastl?lkflpdaqal?kpiriddyee?epilkpgcke?yfwllcklvd?nihikdasqt

1561?tlldldalar?hladcirsre?ildhqdgnve?ddgltgllrl?atsvvkhkpp?fkfsregqef

1621?lrdifnllfl?lpslkdrqqp?kckshssraa?aydllvemvk?gsvenyrlih?nwvmaqhmqs

1681?hapykwdywp?hedvraecrf?vgltnlgatc?ylastiqqly?mipearqavf?takysedmkh

1741?kttllelqkm?ftylmeseck?aynprpfckt?ytmdkqplnt?geqkdmteff?tdlitkieem

1801?spelkntvks?lfggvitnnv?vsldcehvsq?taeefytvrc?qvadmkniye?sldevtikdt

1861?legdnmytcs?qcgkkvraek?racfkklpri?xsfntmrytf?nmvtmmkekv?nthfsfplrl

1921?dmtpytedfl?mgkserkegf?kevsdhskds?esyeydligv?tvhtgtadgg?hyysfirdiv

1981?nphayknnkw?ylfndaevkp?fdsaqlasec?fgqemttkty?dsvtdkfmdf?sfekthsaym

2041?lfykrmepee?engreykfdv?ssellewiwh?dnmqflqdkn?ifehtyfgfm?wqlcscipst

2101?lpdpkavslm?taklstsfvl?etfihskekp?tmlqwiellt?kqfnnsqaac?ewfldrmadd

2161?dwwpmqilik?cpnqivrqmf?qrlcihviqr?lrpvhahlyl?qpgmedgsdd?mdtsvedigg

2221?rscvtrfvrt?lllimehgvk?phskhlteyf?aflyefakmg?eeesqfllsl?qaistmvhfy

2281?mgtkgpenpq?vevlseeegg?eeeeeedils?laeekyrpaa?lekmialval?lveqsrserh

2341?ltlsqtdmaa?ltggkgfpfl?fqhirdgini?rqtcnlifsl?crynnrlaeh?ivsmlftsia

2401?kltpeaanpf?fklltmlmef?aggppgmppf?asyilqriwe?vieynpsqcl?dwlavqtprn

2461?klahswvlqn?menwverfll?ahnyprvrts?aayllvslip?snsfrqmfrs?trslhiptrd

2521?lplspdttvv?lhqvynvllg?llsraklyvd?aavhgttklv?pyfsfmtycl?iskteklmfs

2581?tyfmdlwnlf?qpklsepaia?tnhnkqalls?fwynvcadcp?enirlivqnp?vvtkniafny

2641?iladhddqdv?vlfnrgmlpa?yygilrlcce?qspaftrqla?shqniqwafk?nltphasqyp

2701?gaveelfnlm?qlfiaqrpdm?reeeledikq?fkkttiscyl?rcldgrscwt?tlisafrill

2761?esdedrllvv?fnrglilmte?sfntlhmmyh?eatachvtgd?lvellsifls?vlkstrpylq

2821?rkdvkqaliq?wqeriefahk?lltllnsysp?pelrnacidv?lkelvllsph?dflhtlvpfl

2881?qhnhctyhhs?nipmslgpyf?pcreniklig?gksnirpprp?elnmcllptm?vetskgkddv

2941?ydrmlldyff?syhqfihllc?rvaincekft?etlvklsvlv?ayeglplhla?lfpklwtelc

3001?qtqsamsknc?ikllcedpvf?aeyikcilmd?ertflnnniv?ytfmthfllk?vqsqvfsean

3061?canlistlit?nlisqyqnlq?sdfsnrveis?kasaslngdl?ralalllsvh?tpkqlnpali

3121?ptlqellskc?rtclqqrnsl?qeqeakerkt?kddegatpik?rrrvssdeeh?tvdscisdmk

3181?tetrevltpt?stsdnetrds?siidpgteqd?lpspenssvk?eyrmevpssf?sedmsnirsq

3241?haeeqsnngr?yddckefkdl?hcskdstlas?eesefpstsi?savlsdladl?rscdgqalps

3301?qdpevalsls?cghsrglfsh?mqqhdildtl?crtiestihv?vtrisgkgnq?aas

Table 1 (d)-USP_N01 reference sequences (Derwent AAU82706)

Nucleotide sequence:

atgtgcgaga?actgcgcaga?cctggtggag?gtgttaaatg?aaatatcaga?tgtagaaggt 60

ggtgatggac?tgcagctcag?aaaggaacat?actctcaaaa?tatttactta?catcaattcc 120

tggacacaga?ggcaatgtct?atgctgcttc?aaggaatata?agcatttgga?gatttttaat 180

caagtagtgt?gtgcacttat?taacttagtg?attgcccaag?ttcaagtgct?ccgggaccag 240

ctttgtaaac?attgtactac?cattaacata?gattccacgt?ggcaagatga?gagtaatcaa 300

gcagaagaac?cactgaatat?agatagagag?tgtaatgaag?gaagtacaga?aagacaaaaa 360

tcaatagaaa?aaaaatcaaa?ctctacaaga?atttgtaatc?tgactgagga?ggaatcttca 420

aagagttctg?atccttttag?tttatggagt?acagatgaga?aggaaaaact?cttactatgt 480

gtggcaaaaa?tttttcaaat?tcagtttccc?ttatatactg?cttacaagca?taatactcac 540

cctactattg?aggatatatc?aactcaagaa?agtaacatat?taggggcatt?ctgtgatatg 600

aatgatgtag?aagtaccatt?gcatttgctt?cgttatgtat?gtttgttttg?tgggaaaaat 660

ggcctttctc?tcatgaagga?ttgctttgaa?tatggaactc?ctgaaacttt?gccatttctt 720

atagcacatg?cgtttattac?agttgtgtct?aatattagaa?tatggctaca?tattcccgct 780

gtcatgcagc?acattatacc?ttttaggacc?tatgttatta?ggtatttatg?caagctctcg 840

gatcaggagt?tacgacagag?tgcagctcgt?aacatggctg?acttaatgtg?gagcacagtc 900

aaagaaccat?tggatacaac?attatgcttt?gataaagaaa?gcctagatct?tgcatttaag 960

tactttatgt?cacctacttt?gactatgagg?ttggctggat?tgagtcagat?aacaaatcaa 1020

ctccatacct?tcaatgatgt?gtgcaataat?gaatcattag?tatcggacac?agaaacgtcc 1080

attgcaaaag?aacttgcaga?ctggcttatt?agcaacaatg?tggtggagca?tatatttgga 1140

ccaaatttac?atattgagat?tatcaaacag?tgccaagtga?ttttgaattt?tttggcagca 1200

gaagggcgac?tgagtactca?acatattgac?tgtatttggg?ctgcagcaca?gttgaaacat 1260

tgtagtcggt?atatacatga?cttatttcct?tcactcatca?agaatttgga?tcccgtacca 1320

cttagacatc?tacttaatct?ggtctcagct?cttgagccaa?gtgttcatac?tgaacagaca 1380

ctgtacttgg?catccatgtt?aattaaagca?ctgtggaata?acgcactagc?agctaaggct 1440

cagttatcta?aacagagttc?ttttgcatct?ttattaaata?ctaatattcc?cattggaaat 1500

aagaaagagg?aagaagagct?tagaagaaca?gctccatcac?cttggtcacc?tgcagctagt 1560

cctcaaagca?gtgataatag?cgatacacat?caaagtggag?gtagtgacat?tgaaatggat 1620

gagcaactta?ttaatagaac?caaacatgtg?caacaacgac?tttcagacac?agaggaatcc 1680

atgcagggaa?gttctgacga?aactgccaac?agtggtgaag?atggaagcag?tggtcctggt 1740

agcagtagtg?ggcatagtga?tggatctagc?aatgaggtta?attctagcca?cgcaagccag 1800

tcagctggga?gccctggcag?tgaggtacag?tcagaagaca?ttgcagatat?tgaagccctc 1860

aaagaggaag?atgaagacga?tgatcatggt?cataatcctc?ccaaaagcag?ttgtggtaca 1920

gatcttcgga?atagaaagtt?agagagtcaa?gcaggcattt?gcctggggga?ctcccaaggc 1980

acgtcagaaa?gaaatgggac?aagcagcgga?acaggaaagg?acctggtttt?taacactgaa 2040

tcattgccat?cagtagataa?tcgaatgcga?atgctggatg?cttgttcaca?ctctgaagac 2100

ccagaacatg?atatttcagg?ggaaatgaat?gctactcata?tagcacaagg?gtctcaggag 2160

tcttgtatca?cacgaactgg?ggacttcctt?ggggagacta?ttgggaatga?attatttaat 2220

tgtcgacaat?ttattggtcc?acagcatcac?caccaccacc?accaccatca?ccaccaccac 2280

gatgggcata?tggttgatga?tatgctaagt?gcagatgatg?tcagttgtag?tagctcccag 2340

gttagtgcaa?aatcagaaaa?aaatatggct?gattttgatg?gtgaagaatc?tggatgtgaa 2400

gaggagctag?ttcagattaa?ttcacatgcg?gaactgacat?ctcacctcca?acaacatctt 2460

cccaatttag?cttccattta?ccatgaacat?cttagtcaag?gacctgtagt?tcataaacat 2520

caattcaaca?gtaatgctgt?tacagacatt?aatttggata?atgtttgcaa?gaaaggaaat 2580

actttgttgt?gggatatagt?ccaagatgaa?gatgcagtta?atctttctga?aggattaata 2640

aatgaagcag?agaaacttct?ttgttcgtta?gtatgttggt?ttacagatag?acaaattcga 2700

atgagattca?ttgaaggttg?ccttgaaaac?ttgggaaaca?acagatcagt?agtaatttca 2760

cttcgtcttc?ttccaaaact?atttggtact?tttcagcagt?ttgggagcag?ttacgataca 2820

cactggataa?caatgtgggc?agaaaaagaa?ctgaacatga?tgaagctttt?ctttgataat 2880

ttggtatact?acattcaaac?tgtgagagaa?ggaagacaaa?aacatgcact?gtacagccat 2940

agtgctgaag?ttcaagttcg?tcttcaattc?ttgacttgtg?tattttcaac?tctgggatca 3000

cctgatcatt?tcaggttaag?tttagagcaa?gttgacatct?tatggcattg?tttagtagaa 3060

gattctgaat?gttatgatga?tgcactccat?tggtttttaa?atcaagttcg?aagtaaagat 3120

caacatgcta?tgggtatgga?aacctacaaa?catcttttcc?tggagaagat?gccccagcta 3180

aaacctgaaa?caattagcat?gactggctta?aacctgtttt?cagcatctct?gtaacttggc 3240

tcgattggct?accagtgcct?atgatggttg?ttcaaattct?gagctgtgtg?gtatggacca 3300

attttggggc?attgctttaa?gagcacaatc?tggtgatgtc?agtcgagcag?ctatccagta 3360

tattaactcc?tattatatta?atggtaaaac?aggtttggag?aaggagcaag?aatttattag 3420

taagtgcatg?gagagtctta?tgatagcttc?tagcagtctt?gaacaggaat?cacactcaag 3480

tctcatggtt?atagaaagag?gactccttat?gctgaagaca?catctggaag?cgtttaggag 3540

aaggtttgca?tatcatctga?gacagtggca?aattgaaggc?actggtatta?gtagtcattt 3600

gaaagcactg?agtgacaaac?agtctctgcc?gctaagggtt?gtatgccagc?cagctggact 3660

tcctgacaag?atgactattg?aaatgtatcc?tagtgaccag?gtagcagatc?ttagggctga 3720

agtaactcat?tggtatgaaa?atttacagaa?agaacaaata?aatcaacaag?ctcagcttca 3780

ggagtttggt?caaagcaacc?gaaaaggaga?gtttcctgga?ggcctcatgg?gacctgtcag 3840

gatgatttca?tctggacacg?agttaacaac?agattatgat?gaaaaagcac?ttcatgagct 3900

tggttttaag?gatatgcaga?tggtatttgt?atctttgggt?gcaccaagga?gagagcggaa 3960

aggggaaggt?gttcagctgc?cagcatcttg?cctcccaccc?cctcagaagg?acaacattcc 4020

aatgcttttg?cttttacaag?agcctcattt?aactactctt?tttgatttat?tagagatgct 4080

tgcatcattt?aaaccaccct?caggaaaagt?ggcagtggat?gatagtgaga?gcttacgatg 4140

tgaagaactt?catcttcatg?cagaaaatct?gtctaggcgg?gtctgggagc?tactgatgct 4200

tcttcctaca?tgtcctaata?tgttgatggc?attccagaat?atctcagatg?agcagagttt 4260

taaagctcag?tctgatcaca?ggtctagaca?tgaagtttca?cattattcaa?tgtggctctt 4320

ggtgagttgg?gctcattgct?gttctttagt?gaaatctagc?cttgctgata?gcgatcattt 4380

acaagattgg?ctaaagaaat?tgactctcct?tattcctgag?actgcagttc?gtcatgaatc 4440

atgcagtggt?ctctataagt?tatccctgtc?agggctggat?ggaggagact?caatcaatcg 4500

ttcttttctg?ctattggctg?cctcaacatt?attgaaattt?cttcctgatg?ctcaagcact 4560

caaacctatt?aggatagatg?attatgagga?agaaccaata?ttaaaaccag?gatgtaaaga 4620

gtatttttgg?ttgttatgca?aattagttga?caacatacat?ataaaggacg?ctagtcagac 4680

aacgctcctc?gacttagatg?ccttggcaag?acatttggct?gactgtattc?gaagtaggga 4740

gatccttgat?catcaggatg?gtaatgtaga?agatgatggg?cttacaggac?tcctaaggct 4800

tgcaacaagt?gttgttaaac?acaaaccacc?ctttaaattt?tcaagggaag?gacaggaatt 4860

tttgagagat?atcttcaatc?tcctgttttt?gttgccaagt?ctaaaggacc?gacaacagcc 4920

aaagtgcaaa?tcacattctt?caagagctgc?cgcttacgat?ttgttagtag?agatggtaaa 4980

ggggtctgtt?gagaactaca?ggctaataca?caactgggtt?atggcacaac?acatgcagtc 5040

ccatgcacct?tataaatggg?attactggcc?tcatgaagat?gtccgtgctg?aatgtagatt 5100

tgttggcctt?actaaccttg?gagctacttg?ttacttagct?tctactattc?agcaacttta 5160

tatgatacct?gaggcaagac?aggctgtctt?cactgccaag?tattcagagg?atatgaagca 5220

caagaccact?cttctggagc?ttcagaaaat?gtttacatat?ttaatggaga?gtgaatgcaa 5280

agcatataat?cctagacctt?tctgtaaaac?atacaccatg?gataagcagc?ctctgaatac 5340

tggggaacag?aaagatatga?cagagttttt?tactgatcta?attaccaaaa?tcgaagaaat 5400

gtctcccgaa?ctgaaaaata?ccgtcaaaag?tttatttgga?ggtgtaatta?caaacaatgt 5460

tgtatccttg?gattgtgaac?atgttagtca?aactgctgaa?gagttttata?ctgtgaggtg 5520

ccaagtggct?gatatgaaga?acatttatga?atctcttgat?gaagttacta?taaaagacac 5580

tttggaaggt?gataacatgt?atacttgttc?tcaatgtggg?aagaaagtac?gagctgaaaa 5640

aagggcatgt?tttaagaaat?tgcctcgcat?tttnagtttc?aatactatga?gatacacatt 5700

taatatggtc?acgatgatga?aagagaaagt?gaatacacac?ttttccttcc?cattacgttt 5760

ggacatgacg?ccctatacag?aagattttct?tatgggaaag?agtgagagga?aagaaggttt 5820

taaagaagtc?agtgatcatt?caaaagactc?agagagctat?gaatatgact?tgataggagt 5880

gactgttcac?acaggaacgg?cagatggtgg?acactattat?agctttatca?gagatatagt 5940

aaatccccat?gcttataaaa?acaataaatg?gtatcttttt?aatgatgctg?aggtaaaacc 6000

ttttgattct?gctcaacttg?catctgaatg?ttttggtgga?gagatgacga?ccaagaccta 6060

tgattctgtt?acagataaat?ttatggactt?ctcttttgaa?aagacacaca?gtgcatatat 6120

gctgttttac?aaacgcatgg?aaccagagga?agaaaatggc?agagaataca?aatttgatgt 6180

ttcgtcagag?ttactagagt?ggatttggca?tgataacatg?cagtttcttc?aagacaaaaa 6240

catttttgaa?catacatatt?ttggatttat?gtggcaattg?tgtagttgta?ttcccagtac 6300

attaccagat?cctaaagctg?tgtccttaat?gacagcaaag?ttaagcactt?cctttgtcct 6360

agagacattt?attcattcta?aagaaaagcc?cacgatgctt?cagtggattg?aactgttgac 6420

gaaacagttt?aataatagtc?aggcagcttg?tgagtggttt?ttagatcgta?tggctgatga 6480

cgactggtgg?ccaatgcaga?tactaattaa?gtgccctaat?caaattgtga?gacagatgtt 6540

tcagcgtttg?tgtatccatg?tgattcagag?gctgagacct?gtgcatgctc?atctctattt 6600

gcagccagga?atggaagatg?ggtcagatga?tatggatacc?tcagtagaag?atattggtgg 6660

tcgttcatgt?gtcactcgct?ttgtgagaac?cctgttatta?attatggaac?atggtgtaaa 6720

acctcacagt?aaacatctta?cagagtattt?tgccttcctt?tacgaatttg?caaaaatggg 6780

tgaagaagag?agccaatttt?tgctttcatt?gcaagctata?tctacaatgg?tacattttta 6840

catgggaaca?aaaggacctg?aaaatcctca?agttgaagtg?ttatcagagg?aagaaggggg 6900

agaagaagag?gaggaagaag?atatcctctc?tctggcagaa?gaaaaataca?ggccagctgc 6960

ccttgaaaag?atgatagctt?tagttgctct?tttggttgaa?cagtctcgat?cagaaaggca 7020

tttgacatta?tcacagactg?acatggcagc?attaacagga?ggaaagggat?ttcccttctt 7080

gtttcaacat?attcgtgatg?gcatcaatat?aagacaaact?tgtaatctga?ttttcagcct 7140

gtgtcgatac?aataatcgac?ttgcagaaca?tattgtatct?atgcttttca?catcaatagc 7200

aaagttgact?cctgaggcag?ccaatccttt?ctttaagttg?ttgactatgc?taatggagtt 7260

tgctggtgga?cctccaggaa?tgcctccctt?tgcatcttat?attctgcaga?ggatatggga 7320

ggtgattgaa?tacaatcctt?ctcagtgtct?agattggttg?gcagtgcaga?caccccgaaa 7380

taaactggca?cacagctggg?tcttacagaa?tatggaaaac?tgggtcgagc?ggtttctttt 7440

ggctcacaat?tatcctagag?tgaggacttc?tgcagcttat?cttctggtgt?cccttatacc 7500

aagcaattca?ttccgtcaga?tgttccggtc?aacaaggtct?ttgcacatcc?caacccgtga 7560

ccttccactc?agtccagaca?caacagtagt?cctacatcag?gtctacaacg?tgctccttgg 7620

tttgctctca?agagccaaac?tttatgttga?tgctgctgtt?catggcacta?caaagctagt 7680

gccctatttt?agctttatga?cttactgttt?aatttccaaa?actgagaagc?tgatgttttc 7740

cacatatttc?atggatttgt?ggaacctttt?ccagcctaaa?ctttctgagc?cagcaatagc 7800

tacaaatcac?aataaacagg?ctttgctttc?attttggtac?aatgtctgtg?ctgactgtcc 7860

agagaatatc?cgccttattg?ttcagaaccc?agtggtaacc?aagaacattg?ccttcaatta 7920

catccttgct?gaccatgatg?atcaggatgt?ggtgcttttt?aaccgtggga?tgctgccagc 7980

gtactatggc?attctgaggc?tctgctgtga?gcagtctcct?gcattcacac?gacaactggc 8040

ttctcaccag?aacatccagt?gggcctttaa?gaatcttaca?ccacatgcca?gccaataccc 8100

tggagcagta?gaagaactgt?ttaacctgat?gcagctgttt?atagctcaga?ggccagatat 8160

gagagaagaa?gaattagaag?atattaaaca?gttcaagaaa?acaaccataa?gttgttactt 8220

acgttgctta?gatggccgct?cctgctggac?tactttaata?agtgccttca?gaatactatt 8280

agaatctgat?gaagacagac?ttcttgttgt?atttaatcga?ggattgattc?taatgacaga 8340

gtctttcaac?actttgcaca?tgatgtatca?cgaagctaca?gcttgccatg?tgactggaga 8400

tttagtagaa?cttctgtcaa?tatttctttc?ggttttgaag?tctacacgcc?cttatcttca 8460

gagaaaagat?gtgaaacaag?cattaatcca?gtggcaggag?cgaattgaat?ttgcccataa 8520

actgttaact?cttcttaatt?cctatagtcc?tccagaactt?agaaatgcct?gtatagatgt 8580

cctcaaggaa?cttgtacttt?tgagtcccca?tgattttctt?catactctgg?ttccctttct 8640

acaacacaac?cattgtactt?accatcacag?taatatacca?atgtctcttg?gaccttattt 8700

cccttgtcga?gaaaatatca?agctaatagg?agggaaaagc?aatattcggc?ctccgcgccc 8760

tgaactcaat?atgtgcctct?tgcccacaat?ggtggaaacc?agtaagggca?aagatgacgt 8820

ttatgatcgt?atgctgctag?actacttctt?ttcttatcat?cagttcatcc?atctattatg 8880

ccgagttgca?atcaactgtg?aaaaatttac?tgaaacatta?gttaagctga?gtgtcctagt 8940

tgcctatgaa?ggtttgccac?ttcatcttgc?actgttcccc?aaactttgga?ctgagctatg 9000

ccagactcag?tctgctatgt?caaaaaactg?catcaagctt?ttgtgtgaag?atcctgtttt 9060

cgcagaatat?attaaatgta?tcctaatgga?tgaaagaact?tttttaaaca?acaacattgt 9120

ctacacgttc?atgacacatt?tccttctaaa?ggttcaaagt?caagtgtttt?ctgaagcaaa 9180

ctgtgccaat?ttgatcagca?ctcttattac?aaacttgata?agccagtatc?agaacctaca 9240

gtctgatttc?tccaaccgag?ttgaaatttc?caaagcaagt?gcttctttaa?atggggacct 9300

gagggcactc?gctttgctcc?tgtcagtaca?cactcccaaa?cagttaaacc?cagctctaat 9360

tccaactctg?caagagcttt?taagcaaatg?caggacttgt?ctgcaacaga?gaaactcact 9420

ccaagagcaa?gaagccaaag?aaagaaaaac?taaagatgat?gaaggagcaa?ctcccattaa 9480

aaggcggcgt?gttagcagtg?atgaggagca?cactgtagac?agctgcatca?gtgacatgaa 9540

aacagaaacc?agggaggtcc?tgaccccaac?gagcacttct?gacaatgaga?ccagagactc 9600

ctcaattatt?gatccaggaa?ctgagcaaga?tcttccttcc?cctgaaaata?gttctgttaa 9660

agaataccga?atggaagttc?catcttcgtt?ttcagaagac?atgtcaaata?tcaggtcaca 9720

gcatgcagaa?gaacagtcca?acaatggtag?atatgacgat?tgtaaagaat?ttaaagacct 9780

ccactgttcc?aaggattcta?ccctagctga?ggaagaatct?gagttccctt?ctacttctat 9840

ctctgcagtt?ctgtctgact?tagctgactt?gagaagctgt?gatggccaag?ctttgccctc 9900

ccaggaccct?gaggttgctt?tatctctcag?ttgtggccat?tccagaggac?tctttagtca 9960

tatgcagcaa?catgacattt?tagataccct?gtgtaggacc?attgaatcta?caatccatgt 10020

cgtcacaagg?atatctggca?aaggaaacca?agctgcttct?tga 10063

Table 2 (a) USP_N07: new splice variant-aminoacid sequence

Locus USP_N07 1355aa PRT

Accession number NP_060414

GeneSeq AAU82714

People from source

Insert the splicing form that characterizes with 1:

P0s?14-Pos?81

1 MVPGEENQLV?PKEIENAAEE?PRVLCIIQDT?TNSKTVNERI?TLNLPASTPV?RKLFEDVANK

61 VGYINGTFDL?VWGNGINTAD?MAPLDHTSDK?SLLDANFEPG?KKNFLHLTDK?DGEQPQILLE

121 DSSAGEDSVH?DRFIGPLPRE?GSVGSTSDYV?SQSYSYSSIL?NKSETGYVGL?VNQAMTCYLN

181 SLLQTLFMTP?EFRNALYKWE?FEESEEDPVT?SIPYQLQRLF?VLLQTSKKRA?IETTDVTRSF

241 GWDSSEAWQQ?HDVQELCRVM?FDALEQKWKQ?TEQADLINEL?YQGKLKDYVR?CLECGYEGWR

301 IDTYLDIPLV?IRPYGSSQAF?ASVEEALHAF?IQPEILDGPN?QYFCERCKKK?CDARKGLRFL

361 HFPYLLTLQL?KRFDFDYTTM?HRIKLNDRMT?FPEELDMSTF?IDVEDEKSPQ?TESCTDSGAE

421 NEGSCHSDQM?SNDFSNDDGV?DEGICLETNS?GTEKISKSGL?EKNSLIYELF?SVMVHSGSAA

481 GGHYYACIKS?FSDEQWYSFN?DQHVSRITQE?DIKKTHGGSS?GSRGYYSSAF?ASSTNAYMLI

541 YRLKDPARNA?KFLEVDEYPE?HIKNLVQKER?ELEEQEKRQR?EIERNTCKIK?LFCLHPTKQV

601 MMENKLEVHK?DKTLKEAVEM?AYKMMDLEEV?IPLDCCRLVK?YDEFHDYLER?SYEGEEDTPM

661 GLLLGGVKST?YMFDLLLETR?KPDQVFQSYK?PGEVMVKVHV?VDLKAESVAA?PITVRAYLNQ

721 TVTEFKQLIS?KAIHLPAETM?RIVLERCYND?LRLLSVSSKT?LKAEGFFRSN?KVFVESSETL

781 DYQMAFADSH?LWKLLDRHAN?TIRLFVLLPE?QSPVSYSKRT?AYQKAGGDSG?NVDDDCERVK

841 GPVGSLKSVE?AILEESTEKL?KSLSLQQQQD?GDNGDSSKST?ETSDFENIES?PLNERDSSAS

901 VDNRELEQHI?QTSDPENFQS?EERSDSDVNN?DRSTSSVDSD?ILSSSHSSDT?LCNADNAQIP

961 LANGLDSHSI?TSSRRTKANE?GKKETWDTAE?EDSGTDSEYD?ESGKSRGEMQ?YMYFKAEPYA

1021?ADEGSGEGHK?WLMVHVDKRI?TLAAFKQHLE?PFVGVLSSHF?KVFRVYASNQ?EFESVRLNET

1081?LSSFSDDNKI?TIRLGRALKK?GEYRVKVYQL?LVNEQEPCKF?LLDAVFAKGM?TVRQSKEELI

1141?PQLREQCGLE?LSIDRFRLRK?KTWKNPGTVF?LDYHIYEEDI?NISSNWEVFL?EVLDGVEKMK

1201?SMSQLAVLSR?RWKPSEMKLD?PFQEVVLESS?SVDELREKLS?EISGIPLDDI?EFAKGRGTFP

1261?CDISVLDIHQ?DLDWNPKVST?LNVWPLYICD?DGAVIFYRDK?TEELMELTDE?QRNELMKKES

1321?SRLQKTGHRV?TYSPRKEKAL?KIYLDGAPNK?DLTQD

Table 2 (b) USP_N07: new splice variant-nucleotide sequence

1 atggtgcccg?gcgaggagaa?ccaactggtc?ccgaaagaga?tagaaaatgc?tgctgaagaa

61 cctagagtct?tatgtattat?acaagatact?actaattcaa?agacagtgaa?tgaacggatc

121 actttaaatt?taccagcatc?tactccagtc?agaaagctct?ttgaagatgt?ggccaacaaa

181 gtaggctaca?taaatggaac?ctttgacttg?gtgtggggaa?atggaatcaa?tactgctgat

241 atggcaccac?tggatcatac?cagtgacaag?tcacttctcg?acgctaattt?tgagccagga

301 aagaagaact?ttctgcattt?gacagataaa?gatggtgaac?aacctcaaat?actgctggag

361 gattccagtg?ctggggaaga?cagtgttcat?gacaggttta?taggtccgct?tccaagagaa

421 ggttctgtgg?gttctaccag?tgattatgtc?agccaaagct?actcctactc?atctattttg

481 aataaatcag?aaactggata?tgtgggacta?gtaaaccaag?caatgacttg?ctatttgaat

541 agccttttgc?aaacactttt?tatgactcct?gaatttagga?atgcattata?taagtgggaa

601 tttgaagaat?ctgaagaaga?tccagtgaca?agtattccat?accaacttca?aaggcttttt

661 gttttgttac?aaaccagcaa?aaagagagca?attgaaacca?cagatgttac?aaggagcttt

721 ggatgggata?gtagtgaggc?ttggcagcag?catgatgtac?aagaactatg?cagagtcatg

781 tttgatgctt?tggaacagaa?atggaagcaa?acagaacagg?ctgatcttat?aaatgagcta

841 tatcaaggca?agctgaagga?ctacgtgaga?tgtctggaat?gtggttatga?gggctggcga

901 atcgacacat?atcttgatat?tccattggtc?atccgacctt?atgggtccag?ccaagcattt

961 gctagtgtgg?aagaagcatt?gcatgcattt?attcagccag?agattctgga?tggcccaaat

1021?cagtattttt?gtgaacgttg?taagaagaag?tgtgatgcac?ggaagggcct?tcggtttttg

1081?cattttcctt?atctgctgac?cttacagctg?aaaagattcg?attttgatta?tacaaccatg

1141?cataggatta?aactgaatga?tcgaatgaca?tttcccgagg?aactagatat?gagtactttt

1201?attgatgttg?aagatgagaa?atctcctcag?actgaaagtt?gcactgacag?tggagcagaa

1261?aatgaaggta?gttgtcacag?tgatcagatg?agcaacgatt?tctccaatga?tgatggtgtt

1321?gatgaaggaa?tctgtcttga?aaccaatagt?ggaactgaaa?agatctcaaa?atctggactt

1381?gaaaagaatt?ccttgatcta?tgaacttttc?tctgttatgg?ttcattctgg?gagcgctgct

1441?ggtggtcatt?attatgcatg?tataaagtca?ttcagtgatg?agcagtggta?cagcttcaat

1501?gatcaacatg?tcagcaggat?aacacaagag?gacattaaga?aaacacatgg?tggatcttca

1561?ggaagcagag?gatattattc?tagtgctttc?gcaagttcca?caaatgcata?tatgctgatc

1621?tatagactga?aggatccagc?cagaaatgca?aaatttctag?aagtggatga?atacccagaa

1681?catattaaaa?acttggtgca?gaaagagaga?gagttggaag?aacaagaaaa?gagacaacga

1741?gaaattgagc?gcaatacatg?caagataaaa?ttattctgtt?tgcatcctac?aaaacaagta

1801?atgatggaaa?ataaattgga?ggttcataag?gataagacat?taaaggaagc?agtagaaatg

1861?gcttataaga?tgatggattt?agaagaggta?atacccctgg?attgctgtcg?ccttgttaaa

1921?tatgatgagt?ttcatgatta?tctagaacgg?tcatatgaag?gagaagaaga?tacaccaatg

1981?gggcttctac?taggtggcgt?caagtcaaca?tatatgtttg?atctgctgtt?ggagacgaga

2041?aagcctgatc?aggttttcca?atcttataaa?cctggagaag?tgatggtgaa?agttcatgtt

2101?gttgatctaa?aggcagaatc?tgtagctgct?cctataactg?ttcgtgctta?cttaaatcag

2161?acagttacag?aattcaaaca?actgatttca?aaggccatcc?atttacctgc?tgaaacaatg

2221?agaatagtgc?tggaacgctg?ctacaatgat?ttgcgtcttc?tcagtgtctc?cagtaaaacc

2281?ctgaaagctg?aaggattttt?tagaagtaac?aaggtgtttg?ttgaaagctc?cgagactttg

2341?gattaccaga?tggcctttgc?agactctcat?ttatggaaac?tcctggatcg?gcatgcaaat

2401?acaatcagat?tatttgtttt?gctacctgaa?caatccccag?tatcttattc?caaaaggaca

2461?gcataccaga?aagctggagg?cgattctggt?aatgtggatg?atgactgtga?aagagtcaaa

2521?ggacctgtag?gaagcctaaa?gtctgtggaa?gctattctag?aagaaagcac?tgaaaaactc

2581?aaaagcttgt?cactgcagca?acagcaggat?ggagataatg?gggacagcag?caaaagtact

2641?gagacaagtg?actttgaaaa?catcgaatca?cctctcaatg?agagggactc?ttcagcatca

2701?gtggataata?gagaacttga?acagcatatt?cagacttctg?atccagaaaa?ttttcagtct

2761?gaagaacgat?cagactcaga?tgtgaataat?gacaggagta?caagttcagt?ggacagtgat

2821?attcttagct?ccagtcatag?cagtgatact?ttgtgcaatg?cagacaatgc?tcagatccct

2881?ttggctaatg?gacttgactc?tcacagtatc?acaagtagta?gaagaacgaa?agcaaatgaa

2941?gggaaaaaag?aaacatggga?tacagcagaa?gaagactctg?gaactgatag?tgaatatgat

3001?gagagtggca?agagtagggg?agaaatgcag?tacatgtatt?tcaaagctga?accttatgct

3061?gcagatgaag?gttctgggga?aggacataaa?tggttgatgg?tgcatgttga?taaaagaatt

3121?actctggcag?ctttcaaaca?acatttagag?ccctttgttg?gagttttgtc?ctctcacttc

3181?aaggtctttc?gagtgtatgc?cagcaatcaa?gagtttgaga?gcgtccggct?gaatgagaca

3241?ctttcatcat?tttctgatga?caataagatt?acaattagac?tggggagagc?acttaaaaaa

3301?ggagaataca?gagttaaagt?ataccagctt?ttggtcaatg?aacaagagcc?atgcaagttt

3361?ctgctagatg?ctgtgtttgc?taaaggaatg?actgtacggc?aatcaaaaga?ggaattaatt

3421?cctcagctca?gggagcaatg?tggtttagag?ctcagtattg?acaggtttcg?tctaaggaaa

3481?aaaacatgga?agaatcctgg?cactgtcttt?ttggattatc?atatttatga?agaagatatt

3541?aatatttcca?gcaactggga?ggttttcctt?gaagttcttg?atggggtaga?gaagatgaag

3601?tccatgtcac?agcttgcagt?tttgtcaaga?cggtggaagc?cttcagagat?gaagttggat

3661?cccttccagg?aggttgtatt?ggaaagcagt?agtgtggacg?aattgcgaga?gaagcttagt

3721?gaaatcagtg?ggattccttt?ggatgatatt?gaatttgcta?agggtagagg?aacatttccc

3781?tgtgatattt?ctgtccttga?tattcatcaa?gatttagact?ggaatcctaa?agtttctacc

3841?ctgaatgtct?ggcctcttta?tatctgtgat?gatggtgcgg?tcatatttta?tagggataaa

3901?acagaagaat?taatggaatt?gacagatgag?caaagaaatg?aactgatgaa?aaaagaaagc

3961?agtcgactcc?agaagactgg?acatcgtgta?acatactcac?ctcgtaaaga?gaaagcacta

4021?aaaatatatc?tggatggagc?accaaataaa?gatctgactc?aagactga

Table 2 (c)-USP_N07 reference sequences (Derwent AAU82714)

Aminoacid sequence:

1 mvpgeenqlv?pkeapldhts?dkslldanfe?pgkknflhlt?dkdgeqpqil?ledssageds

61 vhdrfigplp?regsvgstsd?yvsqsysyss?ilnksetgyv?glvnqamtcy?lnsllqtlfm

121 tpefrnalyk?wefeeseedp?vtsipyqlqr?lfvllqtskk?raiettdvtr?sfgwdsseaw

181 qqhdvqelcr?vmfdaleqkw?kqteqadlin?elyqgklkdy?vrclecgyeg?wridtyldip

241 lvirpygssq?afasveealh?afiqpeildg?pnqyfcerck?kkcdarkglr?flhfpylltl

301 qlkrfdfdyt?tmhriklndr?mtfpeeldms?tfidvedeks?pqtesctdsg?aenegschsd

36l qmsndfsndd?gvdegiclet?nsgtekisks?gleknsliye?lfsvmahsgs?aagghyyaci

421 ksfsdeqwys?fddqhvsrit?qedikkthgg?ssgsrgyyss?afasstnaym?liyrlkdpar

481 nakflevgey?pehiknlvqk?ereleeqekr?qreierntck?iklfclhptk?qvmmenklev

541 hkdktlkeav?emaykmmdle?evipldccrl?vkydefhdyl?ersyegeedt?pmglllggvk

601 stymfdllle?trkpdqvfqs?ykpgevmvkv?hvvdlkaesv?aapitvrayl?nqtvtefkql

661 iskaihlpae?tmrivlercy?ndlrllsvss?ktlkaegffr?snkvfvesse?tldyqmafad

721 shlwklldrh?antirlfvll?peqspvsysk?rtayqkaggd?sgnvdddcer?vkgpvgslks

781 veaileeste?klkslslqqq?qdgdngdssk?stetsdfeni?esplnerdss?asvdnreleq

841 hiqtsdpenf?qseersdsdv?nndrstssvd?sdilssshss?dtlcnadnaq?iplangldsh

901 sitssrrtka?negkketwdt?aeedsgtdse?ydesgksrge?mqymyfkaep?yaadegsgeg

961 hkwlmvhvdk?ritlaafkqh?lepfvgvlss?hfkvfrvyas?nqefesvrln?etlssfsddn

1021?kitirlgral?kkgeyrvkvy?qllvneqepc?kflldavfak?gmtvrqskee?lipqlreqcg

1081?lelsidrfrl?rkktwknpgt?vfldyhiyee?dinissnwev?flevldgvek?mksmsqlavl

1141?srrwkpsemk?ldpfqevvle?sssvdelrek?lseisgipld?diefakgrgt?fpcdisvldi

1201?hqdldwnpkv?stlnvwplyi?cddgavifyr?dkteelmelt?deqrnelmkk?essrlqktgh

1261?rvtysprkek?alkiyldgap?nkdltqd

Table 2 (d)-USP_N07 reference sequences (Derwent AAU82714)

Nucleotide sequence:

atggtgcccg?gcgaggagaa?ccaactggtc?ccgaaagagg?caccactgga?tcataccagt 60

gacaagtcac?ttctcgacgc?taattttgag?ccaggaaaga?agaactttct?gcatttgaca 120

gataaagatg?gtgaacaacc?tcaaatactg?ctggaggatt?ccagtgctgg?ggaagacagt 180

gttcatgaca?ggtttatagg?tccgcttcca?agagaaggtt?ctgtgggttc?taccagtgat 240

tatgtcagcc?aaagctactc?ctactcatct?attttgaata?aatcagaaac?tggatatgtg 300

ggactagtaa?accaagcaat?gacttgctat?ttgaatagcc?ttttgcaaac?actttttatg 360

actcctgaat?ttaggaatgc?attatataag?tgggaatttg?aagaatctga?agaagatcca 420

gtgacaagta?ttccatacca?acttcaaagg?ctttttgttt?tgttacaaac?cagcaaaaag 480

agagcaattg?aaaccacaga?tgttacaagg?agctttggat?gggatagtag?tgaggcttgg 540

cagcagcatg?atgtacaaga?actatgcaga?gtcatgtttg?atgctttgga?acagaaatgg 600

aagcaaacag?aacaggctga?tcttataaat?gagctatatc?aaggcaagct?gaaggactac 660

gtgagatgtc?tggaatgtgg?ttatgagggc?tggcgaatcg?acacatatct?tgatatccca 720

ttggtcatcc?gaccttatgg?gtccagccaa?gcatttgcta?gtgtggaaga?agcattgcat 780

gcatttattc?agccagagat?tctggatggc?ccaaatcagt?atttttgtga?acgttgtaag 840

aagaagtgtg?atgcacggaa?gggccttcgg?tttttgcatt?ttccttatct?gctgacctta 900

cagctgaaaa?gattcgattt?tgattataca?accatgcata?ggattaaact?gaatgatcga 960

atgacatttc?ccgaggaact?agatatgagt?acttttattg?atgttgaaga?tgagaaatct 1020

cctcagactg?aaagttgcac?tgacagtgga?gcagaaaatg?aaggtagttg?tcacagtgat 1080

cagatgagca?acgatttctc?caatgatgat?ggtgttgatg?aaggaatctg?tcttgaaacc 1140

aatagtggaa?ctgaaaagat?ctcaaaatct?ggacttgaaa?agaattcctt?gatctatgaa 1200

cttttctctg?ttatggctca?ttctgggagc?gctgctggtg?gtcattatta?tgcatgtata 1260

aagtcattca?gtgatgagca?gtggtacagc?ttcgatgatc?aacatgtcag?caggataaca 1320

caagaggaca?ttaagaaaac?acatggtgga?tcttcaggaa?gcagaggata?ttattctagt 1380

gctttcgcaa?gttccacaaa?tgcatatatg?ctgatctata?gactgaagga?tccagccaga 1440

aatgcaaaat?ttctagaagt?gggtgaatac?ccagaacata?ttaaaaactt?ggtgcagaaa 1500

gagagagagt?tggaagaaca?agaaaagaga?caacgagaaa?ttgagcgcaa?tacatgcaag 1560

ataaaattat?tctgtttgca?tcctacaaaa?caagtaatga?tggaaaataa?attggaggtt 1620

cataaggata?agacattaaa?ggaagcagta?gaaatggctt?ataagatgat?ggatttagaa 1680

gaggtaatac?ccctggattg?ctgtcgcctt?gttaaatatg?atgagtttca?tgattatcta 1740

gaacggtcat?atgaaggaga?agaagataca?ccaatggggc?ttctactagg?tggcgtcaag 1800

tcaacatata?tgtttgatct?gctgttggag?acgagaaagc?ctgatcaggt?tttccaatct 1860

tataaacctg?gagaagtgat?ggtgaaagtt?catgttgttg?atctaaaggc?agaatctgta 1920

gctgctccta?taactgttcg?tgcttactta?aatcagacag?ttacagaatt?caaacaactg 1980

atttcaaagg?ccatccattt?acctgctgaa?acaatgagaa?tagtgctgga?acgctgctac 2040

aatgatttgc?gtcttctcag?tgtctccagt?aaaaccctga?aagctgaagg?attttttaga 2100

agtaacaagg?tgtttgttga?aagctccgag?actttggatt?accagatggc?ctttgcagac 2160

tctcatttat?ggaaactcct?ggatcggcat?gcaaatacaa?tcagattatt?tgttttgcta 2220

cctgaacaat?ccccagtatc?ttattccaaa?aggacagcat?accagaaagc?tggaggcgat 2280

tctggtaatg?tggatgatga?ctgtgaaaga?gtcaaaggac?ctgtaggaag?cctaaagtct 2340

gtggaagcta?ttctagaaga?aagcactgaa?aaactcaaaa?gcttgtcact?gcagcaacag 2400

caggatggag?ataatgggga?cagcagcaaa?agtactgaga?caagtgactt?tgaaaacatc 2460

gaatcacctc?tcaatgagag?ggactcttca?gcatcagtgg?ataatagaga?acttgaacag 2520

catattcaga?cttctgatcc?agaaaatttt?cagtctgaag?aacgatcaga?ctcagatgtg 2580

aataatgaca?ggagtacaag?ttcagtggac?agtgatattc?ttagctccag?tcatagcagt 2640

gatactttgt?gcaatgcaga?caatgctcag?atccctttgg?ctaatggact?tgactctcac 2700

agtatcacaa?gtagtagaag?aacgaaagca?aatgaaggga?aaaaagaaac?atgggataca 2760

gcagaagaag?actctggaac?tgatagtgaa?tatgatgaga?gtggcaagag?taggggagaa 2820

atgcagtaca?tgtatttcaa?agctgaacct?tatgctgcag?atgaaggttc?tggggaagga 2880

cataaatggt?tgatggtgca?tgttgataaa?agaattactc?tggcagcttt?caaacaacat 2940

ttagagccct?ttgttggagt?tttgtcctct?cacttcaagg?tctttcgagt?gtatgccagc 3000

aatcaagagt?ttgagagcgt?ccggctgaat?gagacacttt?catcattttc?tgatgacaat 3060

aagattacaa?ttagactggg?gagagcactt?aaaaaaggag?aatacagagt?taaagtatac 3120

cagcttttgg?tcaatgaaca?agagccatgc?aagtttctgc?tagatgctgt?gtttgctaaa 3180

ggaatgactg?tacggcaatc?aaaagaggaa?ttaattcctc?agctcaggga?gcaatgtggt 3240

ttagagctca?gtattgacag?gtttcgtcta?aggaaaaaaa?catggaagaa?tcctggcact 3300

gtctttttgg?attatcatat?ttatgaagaa?gatattaata?tttccagcaa?ctgggaggtt 3360

ttccttgaag?ttcttgatgg?ggtagagaag?atgaagtcca?tgtcacagct?tgcagttttg 3420

tcaagacggt?ggaagccttc?agagatgaag?ttggatccct?tccaggaggt?tgtattggaa 3480

agcagtagtg?tggacgaatt?gcgagagaag?cttagtgaaa?tcagtgggat?tcctttggat 3540

gatattgaat?ttgctaaggg?tagaggaaca?tttccctgtg?atatttctgt?ccttgatatt 3600

catcaggatt?tagactggaa?tcctaaagtt?tctaccctga?atgtctggcc?tctttatatc 3660

tgtgatgatg?gtgcggtcat?attttatagg?gataaaacag?aagaattaat?ggaattgaca 3720

gatgagcaaa?gaaatgaact?gatgaaaaaa?gaaagcagtc?gactccagaa?gactggacat 3780

cgtgtaacat?actcacctcg?taaagagaaa?gcactaaaaa?tatatctgga?tggagcacca 3840

aataaagatc?tgactcaaga?ctga 3864

Table 3 (a)-USP_N11: new splice variant: aminoacid sequence

Locus USP_N11 402aa PRT

Accession number AK022614

GeneSeq AAU82713

People from source

Insert the splicing form that characterizes with 1:

Pos?12-Pos?48

1 MTVRNIASIC?NMEEPPALGS?PGWTLLAPPL?VRAFGELRLE?EGIAVPCRGT?NASALEKDIG

61 PEQFPINEHY?FGLVNFGNTC?YCNSVLQALY?FCRPFRENVL?AYKAQQKKKE?NLLTCLADLF

121 HSIATQKKKV?GVIPPKKFIS?RLRKENDLFD?NYMQQDAHEF?LNYLLNTIAD?ILQEEKKQEK

181 QNGKLKNGNM?NEPAENNKPE?LTWVHEIFQG?TLTNETRCLN?CETVSSKDED?FLDLSVDVEQ

241 NTSITHCLRD?FSNTETLCSE?QKYYCETCCS?KQEAQKRMRV?KKLPMILALH?LKRFKYMEQL

301 HRYTKLSYRV?VFPLELRLFN?TSSDAVNLDR?MYDLVAVVVH?CGSGPNRGHY?ITIVKSHGFW

361 LLFDDDIVEK?IDAQAIEEFY?GLTSDISKNS?ESGYILFYQS?RE

Table 3 (b)-USP_N11: new splice variant: nucleotide sequence

1 atgactgtcc?gaaacatcgc?ctccatctgt?aatatgggca?ccaatgcctc?tgctctggaa

61 aaagacattg?gtccagagca?gtttccaatc?aatgaacact?atttcggatt?ggtcaatttt

121 ggaaacacat?gctactgtaa?ctccgtgctt?caggcattgt?acttctgccg?tccattccgg

181 gagaatgtgt?tggcatacaa?ggcccagcaa?aagaagaagg?aaaacttgct?gacgtgcctg

241 gcggaccttt?tccacagcat?tgccacacag?aagaagaagg?ttggcgtcat?cccaccaaag

301 aagttcattt?caaggctgag?aaaagagaat?gatctctttg?ataactacat?gcagcaggat

361 gctcatgaat?ttttaaatta?tttgctaaac?actattgcgg?acatccttca?ggaggagaag

421 aaacaggaaa?aacaaaatgg?aaaattaaaa?aatggcaaca?tgaacgaacc?tgcggaaaat

481 aataaaccag?aactcacctg?ggtccatgag?atttttcagg?gaacgcttac?caatgaaact

541 cgatgcttga?actgtgaaac?tgttagtagc?aaagatgaag?attttcttga?cctttctgtt

601 gatgtggagc?agaatacatc?cattacccac?tgtctaagag?acttcagcaa?cacagaaaca

661 ctgtgtagtg?aacaaaaata?ttattgtgaa?acatgctgca?gcaaacaaga?agcccagaaa

721 aggatgaggg?taaaaaagct?gcccatgatc?ttggccctgc?acctaaagcg?gttcaagtac

781 atggagcagc?tgcacagata?caccaagctg?tcttaccgtg?tggtcttccc?tctggaactc

841 cggctcttca?acacctccag?tgatgcagtg?aacctggacc?gcatgtatga?cttggttgcg

901 gtggtcgttc?actgtggcag?tggtcctaat?cgtgggcatt?atatcactat?tgtgaaaagt

961 cacggcttct?ggcttttgtt?tgatgatgac?attgtagaga?aaatagatgc?tcaagctatt

1021?gaagaattct?atggcctgac?gtcagatata?tcaaaaaatt?cagaatctgg?atatatttta

1081?ttctatcagt?caagagagta?a

Table 3 (c)-USP_N11 reference sequences (Derwent AAU82713)

Aminoacid sequence:

1 mtvrniasic?nmgtnasale?kdigpeqfpi?nehyfglvnf?gntcycnsvl?qalyfcrpfr

61 envlaykaqq?kkkenlltcl?adlfhsiatq?kkkvgvippk?kfisrlrken?dlfdnymqqd

121 aheflnylln?tiadilqeek?kqekqngklk?ngnmnepaen?nkpeltwvhe?ifqgtltnet

181 rclncetvss?kdedfldlsv?dveqntsith?clrdfsntet?lcseqkyyce?tccskqeaqk

241 rmrvkklpmv?lalhlkrfky?meqlrrytkl?syrvvfplel?rlfntssdav?nldrmydlva

301 vvvhcgsgpn?rghyitivks?hgfwllfddd?ivekidaqai?eefygltsdi?sknsesgyil

361 fyqsre

Table 3 (d)-USP_N11 reference sequences (Derwent AAU82713)

Nucleotide sequence:

atgactgtcc?gaaacatcgc?ctccatctgt?aatatgggca?ccaatgcctc?tgctctggaa 60

aaagacattg?gtccagagca?gtttccaatc?aatgaacact?atttcggatt?ggtcaatttt 120

ggaaacacat?gctactgtaa?ctccgtgctt?caggcattgt?acttctgccg?tccattccgg 180

gagaatgtgt?tggcatacaa?ggcccagcaa?aagaagaagg?aaaacttgct?gacgtgcctg 240

gcggaccttt?tccacagcat?tgccacacag?aagaagaagg?ttggcgtcat?cccaccaaag 300

aagttcattt?caaggctgag?aaaagagaat?gatctctttg?ataactacat?gcagcaggat 360

gctcatgaat?ttttaaatta?tttgctaaac?actattgcgg?acatccttca?ggaggagaag 420

aaacaggaaa?aacaaaatgg?aaaattaaaa?aatggcaaca?tgaacgaacc?tgcggaaaat 480

aataaaccag?aactcacctg?ggtccatgag?atttttcagg?gaacgcttac?caatgaaact 540

cgatgcttga?actgtgaaac?tgttagtagc?aaagatgaag?attttcttga?cctttctgtt 600

gatgtggagc?agaatacatc?cattacccac?tgtctaagag?acttcagcaa?cacagaaaca 660

ctgtgtagtg?aacaaaaata?ttattgtgaa?acatgctgca?gcaaacaaga?agcccagaaa 720

aggatgaggg?taaaaaagct?gcccatggtc?ttggccctgc?acctaaagcg?gttcaagtac 780

atggagcagc?tgcgcagata?caccaagctg?tcttaccgtg?tggtcttccc?tctggaactc 840

cggctcttca?acacctccag?tgatgcagtg?aacctggacc?gcatgtatga?cttggttgcg 900

gtggtcgttc?actgtggcag?tggtcctaat?cgtgggcatt?atatcactat?tgtgaaaagt 960

cacggcttct?ggcttttgtt?tgatgatgac?attgtagaga?aaatagatgc?tcaagctatt 1020

gaagaattct?atggcctgac?gtcagatata?tcaaaaaatt?cagaatctgg?atatatttta 1080

ttctatcagt?caagagagta?a 1101

Embodiment

The explicitly known family member of the C12/C19 of CA/ family of family (taking off the ubiquitin enzyme) retrieves (C12 has 9 peptide sequences and C19 has 76 peptide sequences) and they is carried out PSI-BLAST and Smith-Waterman search from MEROPS.Search database comprises the protein of the open reading-frame (ORF) sequence of coming from the translation of Celera and public (NCBI) human genomic sequence, Celera prediction, based on the Celera genescan prediction of Celera human genome with from the forecasting sequence of right of ownership database.The member of two families (C12 and C19) searches for to identify their Interpro motif through Interproscan in addition.All are identified with relevant Prints, Prosite and PFAM pattern (PR00707, PS00140 and PF01088) by IPR001578 from the protein of C12 family (ubiquitin carboxyl terminal lytic enzyme, family 1).Similarly, the C19 of family is by IPR001394 motif (ubiquitin thioesterase) and corresponding Prints and PFAM label (PS00972, PS00973, PS50235, PF00443) definition.To download and be used for suitable search from the model of Prosite, Prints and PFAM database.Gather and repeat to reduce from the result of multiple search by three kinds of filters processing.With the elementary result of meeting according to low-complexity screening and with the protein reference database coupling that contains from the human protein sequence of GenBank, SwissProt and Refseq.Shift out with reference database in protein at least 50 amino acid, have the result that meets greater than 95% identity.In addition, the corresponding DNA sequence and the DNA reference database coupling that contains the human cDNA sequence of Refseq that these are met the result.Shift out with reference database in dna sequence dna at least 150 Nucleotide, have sequence greater than 95% identity.And use 95% identity boundary to make that the remaining result of meeting removes mutually to produce the colony that the result forms that meets by high homology from filter method at last.USP_N01 is that the representativeness from this kind of groups meets sequence.

Everyone class members of the C12/C19 of family is carried out second take turns that to analyze be that gene expression profile and electronics Northern analyze.C12/C19 family member's common sequence is used to identify corresponding UniGene colony and relevant standardization body's type expression and distribution.These are analyzed with corresponding probe is provided with the gene chip data comparison that is obtained on human tissue collection of illustrative plates and the tumor sample by searching.

Embodiment 1

Gene expression profile corresponding to the USP_N01 of SEQ ID.No.1 and SEQ ID.No.2 shows at Fig. 1.

Embodiment 2

Gene expression profile corresponding to the USP_N07 of SEQ ID.No.5 and SEQ ID.No.6 shows at Fig. 2.

Embodiment 3

Gene expression profile corresponding to the USP_N11 of SEQ ID.No.9 and SEQ ID.No.10 shows at Fig. 3.

Claims (32)

1. separated DNA, it comprises the nucleotide sequence that is selected from following coding ubiquitin specific protease:

(a)SEQ?ID?No.2、SEQ?ID?No.6、SEQ.ID?No.10；

(b) fragment of SEQ ID No.2, SEQ ID No.6, SEQ.ID No.10;

(c) derivative of SEQ ID No.2, SEQ ID No.6, SEQ.ID No.10;

(d) with SEQ ID No.2, the basic homologous sequence of SEQ ID No.6, SEQ.ID No.10.

2. separated DNA according to claim 1, it is made up of the nucleotide sequence that is selected from SEQ ID No.2, SEQ ID No.6 or SEQ.ID No.10.

3. separated DNA according to claim 1, wherein said fragment comprises at least 50 Nucleotide in the nucleotide sequence that is selected from SEQID No.2, SEQ ID No.6 or SEQ.ID No.10 and has kept ubiquitin specific function activity.

4. separated DNA according to claim 1, it comprises and is selected from nucleotide sequence at least 80% homology of SEQ ID No.2, SEQ ID No.6 or SEQ.ID No.10 and has kept the active nucleotide sequence of ubiquitin specific function.

5. separated DNA, its be included under the high stringent condition with claim 1-4 in the nucleotide sequence of any described about 30-50 Nucleotide through DNA isolation hybridization.

6. the separated polypeptide of ubiquitin specific protease, it comprises and is selected from following aminoacid sequence:

(a)SEQ.ID.No.1、SEQ.ID.No.5、SEQ.ID?No.9；

(b) fragment of SEQ.ID.No.1, SEQ.ID.No.5, SEQ.ID No.9;

(c) derivative of SEQ.ID.No.1, SEQ.ID.No.5, SEQ.ID No.9;

(d) with SEQ.ID.No.1, SEQ.ID.No.5, the basic homologous sequence of SEQ.ID No.9.

7. separated polypeptide according to claim 6, it is made up of the aminoacid sequence that is selected from SEQ ID No.1, SEQ ID No.5 or SEQ.ID No.9.

8. separated polypeptide according to claim 6, wherein said fragment comprise at least 5 successive amino acid in the aminoacid sequence that is selected from SEQID No.1, SEQ ID No.5 or SEQ.ID No.9.

9. separated polypeptide according to claim 6, its be selected from aminoacid sequence at least 80% homology of SEQ ID No.1, SEQ ID No.5 or SEQ.ID No.9 and kept ubiquitin specific function activity.

10. separated polypeptide according to claim 6, wherein said derivative are the function equivalents of ubiquitin specific protease.

11. host cell, it comprises according to any described separated DNA among the claim 1-5.

12. carrier, it comprises according to any described separated DNA among the claim 1-5.

13. carrier according to claim 12, it comprises transcriptional control sequence.

14. host cell, it comprises according to claim 12 or 13 described carriers.

15. be used for the method for diagnosing human mammary cancer, it comprises that mensuration is from the polypeptide amount according to any described polypeptide among the claim 6-10 of comprising in people's mammary tissue, wherein for the amount of polypeptide described in the normal galactophore tissue, the amount that described polypeptide raises suffers from mammary cancer for described people and has diagnostic significance.

16. according to the diagnostic method of claim 15, wherein said detection step comprises described mammary tissue contacted with such antibody, described antibody with comprise claim 6-10 in the polypeptid specificity of any described aminoacid sequence combine; And detect and to combine with the specificity of polypeptide at antibody described in the described mammary tissue, wherein detect specificity to polypeptide in conjunction with showing that existence comprises the polypeptide of any described aminoacid sequence among the claim 6-10.

17. according to the diagnostic method of claim 15 or claim 16, wherein said aminoacid sequence comprises SEQ ID.No.1, its fragment, its derivative or its homologue.

18. be used to diagnose the method for human leukemia, it comprises especially the polypeptide amount according to any described polypeptide among the claim 6-10 of comprising in the lymphoidocyte of peripheral blood cells of measuring, wherein for the amount of normal circumference hemocyte especially polypeptide described in the lymphoidocyte, the amount that described polypeptide raises suffers from leukemia for described people and has diagnostic significance.

19. the described method of claim 18, wherein said detection step comprises described peripheral blood cells especially lymphoidocyte contacted with such antibody, described antibody with comprise claim 6-10 in the polypeptid specificity of any described aminoacid sequence combine; And detect and to combine with the specificity of polypeptide in described peripheral blood cells especially antibody described in the lymphoidocyte, wherein detect specificity to polypeptide in conjunction with showing that existence comprises the polypeptide of any described aminoacid sequence among the claim 6-10.

20. according to the diagnostic method of claim 18 or claim 19, wherein said aminoacid sequence comprises SEQ ID.No.5, its fragment, its derivative or its homologue.

21. be used for the method for diagnosing human functional disorders of brain, it comprises the polypeptide amount of measuring in amygdala, spinal cord or the olfactory bulb tissue according to any described polypeptide among the claim 6-10 of comprising, wherein for the amount of polypeptide described in amygdala, spinal cord and the olfactory bulb tissue, the amount that described polypeptide raises suffers from functional disorders of brain for described people and has diagnostic significance.

22. the described method of claim 21, wherein said detection step comprise described amygdala, spinal cord or olfactory bulb tissue are contacted with such antibody, described antibody with comprise claim 6-10 in the polypeptid specificity of any described aminoacid sequence combine; And detect and to combine with the specificity of polypeptide at antibody described in the described cerebral tissue, wherein detect specificity to polypeptide in conjunction with showing that existence comprises the polypeptide of any described aminoacid sequence among the claim 6-10.

23. according to the diagnostic method of claim 21 or claim 22, wherein said aminoacid sequence comprises SEQ ID.No.9, its fragment, its derivative or its homologue.

24. antibody, its specificity is in conjunction with the polypeptide that comprises any described aminoacid sequence among the claim 6-10.

25. antibody according to claim 24, it is Fab or (Fab ') 2 fragments.

26. antibody according to claim 24, it is a polyclonal antibody.

27. antibody according to claim 24, it is a monoclonal antibody.

28. pharmaceutical composition, it comprises the described antibody according to claim 24-27.

29. be used for the treatment of the method for mammary cancer, it comprises inhibition nucleic acid from the genetic expression that is suitable for suppressing such of significant quantity to the patient with breast cancer that use, described gene comprises the nucleotide sequence that is selected from SEQ ID No.2, SEQID No.6, SEQ.ID No.10.

30. be used for the treatment of leukemic method, it comprises inhibition nucleic acid from the genetic expression that is suitable for suppressing such of significant quantity to the leukaemic that use, described gene comprises the nucleotide sequence that is selected from SEQ ID No.2, SEQID No.6, SEQ.ID No.10.

31. pharmaceutical composition, it comprises inhibition nucleic acid and pharmaceutically acceptable carrier of the genetic expression that is suitable for suppressing such, and wherein said gene comprises the nucleotide sequence that is selected from SEQ ID No.2, SEQ ID No.6, SEQ.IDNo.10.

32. be used to relate to the diagnostic kit of the disease of ubiquitin specific protease, it comprises at least a following component:

(a) oligonucleotide, it is suitable for detecting the nucleic acid that comprises shown in SEQ ID No.2, SEQ ID.No.6 or SEQID No.10 nucleotide sequence or its part,

(b) antibody, it is suitable for detecting the polypeptide that comprises shown in SEQ ID No.1, SEQ ID.No.5 or SEQ IDNo.9 aminoacid sequence or its part,

(c) working instructions of described test kit.

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