CA2245242C - Purified sr-p70 protein - Google Patents

Abstract

Novel nucleic acid sequences from the tumour-suppressor gene family related to the gene of protein p53, and the corresponding protein sequences, are disclosed.

Description

WO 97/28186 PCT ~ R97 / 00214 "Protein puri ~ iee SR-p70".
The invention relates to novel nucleic acid sequences of the family of tumor suppressor genes related to the p53 gene, and the corresponding protein sequences.
The invention also relates to prophylactic, therapeutic and ,,,, 5 diagnostics of these, particularly in the field of pathologies related to Phenomena of apoplosis or cell death.
Tumor suppressor genes play a key role in protecting against cancer phenomena, and any modi ~ i ~, alion likely to cause the loss of one of these genes, its inactivation or dysfunction, may have a oncogenic character, thus creating favorable conditions for the development of a Cancer.
The authors of the present invention have identified the transcripts of a new gene as well as the corresponding proteins. This SR-p70 gene is related to the tumor suppressor gene p53, whose anti-tumor activity is related to its activity as a factor in the control and more specifically to the controls exercised on the activity of the Bax and Bc1-2 genes, instrumental in cell death mechanisms.
The present invention therefore relates to purified SR-p70 proteins, or biologically active fragments thereof.
The invention also provides isolated nucleic acid sequences encoding for said poii.les or their biologically active fragments and specific oligonucleotides obtained from these sequences.
It also targets the expression cloning vectors eVou containing at least one of the nurteuretic sequences defined above, and the hepatocytes l, ansrec-by these cloning and / or expression vectors under conditions the l ~ pl; c ~ lioll e ~ t / or the expression of one of the ~ it ~ s sec ~ uence nucleotide Methods of producing proteins identified as SR-p70 or their fragments B cells are actively acti ~ ed by the transfected host cells is also included of the invention.
The invention also includes antibodies or specific antibody derivatives Derri proteins. ss above, It also aims at detecting cancer, either by measuring the accumulation of SR-p70 proteins in tumors according to Immune l ~ iaLochi. "a, either by highlighting in the patients' senum to self-release ~, s directed against these proteins.

WO 97/28186 PCT ~ FR97100214 The invention also relates to any inhibitor or activator of the activity of SR-p70 for example protein-protein interaction involving SR-p70.
It also relates to antisense oligonucleotide sequences, specific for nucleic acid sequences above, capable of modulating the expression of SR-p70 gene in vivo.
The invention finally comprises a method of gene therapy in which vectors such as, for example, inactivated viral vectors capable of transferring coding sequences for a protein according to the invention are injected into cells deficient for this protein, for purposes of regulating the phenomena 0 of apoptosis or reversion of immunity.
The subject of the present invention is a purified polypeptide comprising a sequence amino acid selected from:
a) the sequence SEQ ID n ~ 2;
b ~ the sequence SEQ ID n ~ 4;
c) the sequence SEQ ID n ~ 6;
d) the sequence SEQ ID n ~ 8;
e) the sequence SEQ ID n ~ 10;
f) the sequence SEQ ID No. 13;
g) the sequence SEQ ID No. 15;
h) the sequence SEQ ID No. 17;
i) the sequence SEQ ID No. 19;
j) any biologically active sequence derived from SEQ ID No. 2, SEQ ID No. 4, SEQ IDN ~ 6, SEQ IDN ~ 8, SEQ IDN ~ 10, SEQ IDN ~ 13, SEQ IDN ~ 15, SEQ IDN ~ 17 or SEQ II: ~ n ~ 19.
In the ~ iG ~ of the invention, the following are used:
- Pr ~ lei., e SR-p70: a poly ~ eplide comprising a sequence of amino acids chosen from the sequences SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEC, ID No. 8, SEQ ID No. 1u, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17 or SEQ ID No. 19, or any biologically active fragment or derivative thereof.
derivative: any variant polypeptide of the polypeptide of sequence SEQ ID No. 2, SEQ
ID n ~ 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17 or SEQ ID No. 19, or any molecule resulting from an odiriacy of nature genetics eUou cl ~ ue sequence SEQ ID n ~ 2, SEQ ID n ~ 4, SEQ ID n ~ 6, SEQ ID n ~ 8, SEQ ID n ~ 1 0, SEQ ID n ~ 1 3, SEQ ID n ~ 1 ~, SEQ ID n ~ 1 7 or SEQ ID n ~
3 ~ 19 i.e. obtained by mutation, deletion, addition, substitution eVou modification of a single or a limited number of amino acids, as well as any WO 97/28186 PCTA ~ R97 / 00214 -isoform that is to say a sequence identical to the sequence SEQ ID n ~ ~ SEQ iD
n ~ 4 SEQ ID No ~ 6 SEQ ID No ~ 8 SEQ ID No ~ 10 SEQ ID No ~ 13 SEQ ID No ~ 15 SEQ ID
- n ~ 17 or SEQ ID n ~ 19 to one of his ~ ragments or modified sequences contained one or more amino acids in the enantiomeric form D, said sequences modified variants or isoforms having at least one of the properties making it biologically active.
- biologically active: c ~ p ~ hle to bind to eVou DNA to exert an activity of transcription factor eVou pa, participate in conl, ole the cell cycle of the diffel ~ nialion and apoptosis eVou c ~ pa ~ IQ to be recognized by antico "~ s specific to the sequence polypeptide SEQ ID No. 2 SEC2 ID No. 4 SEQ ID No. 6 SEQ
ID No. 8 SEQ ID No. 10 SEQ ID No. 13 SEQ ID No. 15 SEQ ID No. 17 or SEQ ID No. 19 eVou capable of inducing antibodies that recognize this polypeptide.
The manufacture of derivatives can have dirterenla objectives including in particular that to increase the affinity of the polypeptide for DNA or its transc, -iption that of improving its prosiuction rates of au ~ ", en, 'er its resiaLc" ~ ce proteases from, or to change its biological activities or to confer new pl ~ al "~ aceutiques and / or biolag properties ~ ~ ues Among the polypeptides of the invention preferred pol ~ "~ eplide of human origin ## EQU1 ## SEQ ID ## EQU1 ## SEQ ID ## EQU1 ##
SEQ ID No. 19. The 636 amino acid polypeptide corresponding to the sequence SEQ ID No. 6 is more than 97% identical to the polypeptide of sequence SEQ ID No. 2. The polypeptide of sequence SEQ ID No. 2 and that of sequence SEQ ID No. 4 are two.
expression products of the same gene for the same sequences SEQ ID n ~ 8 and SEQ ID n ~ 10 and for the sequences SEQ ID n ~ 6 SEQ ID n ~ 13 SEQ ID n ~ 15 SEQ
~ ~ I Dn 17etSE.QlDn 19.
As will be explained in the examples, the polypeptide of sequence SEQ ID No. 4 corresponds to a t ~ i ", premature inaisol1 sequence peptide sequence SEQ ID n ~ 2 to a splice ally ~ ~ alit of the llanscr, pt coding for the polypeptide of SEQ ID n ~ 2 the longer (messenger RNA) gene c ~ "esponda" i. Similarly in humans cGI ~ polypeptides responding to the sequences SEQ ID n ~ 6 SEQ ID No. 13 SEQ ID No. 15 SEQ ID No. 17 and SEQ ID No. 19 differ in their composition at the level of the parts N- eVou -C-terr, -, inals and this consecutive to splices ~ al, "alirs of the same The age is primary. The N-terminal peptide sequence of the sequence SEQ ID n ~
10 is deleted this linked to an alternative splicing of its Llal1sc ~ i ~, t coding Advantageously, the invention aims at a CGI polypeptide, responding to the DNA binding of one of the above polypeptides.

WO 97/28186 PCTA ~ R97 / 00214 This domain corresponds to the sequence between the residue 110 and the residue 310 for the sequences SEQ ID n ~ 2 or 6, and between the residue 60 and the residue 260 for the sequence SEQ ID n ~ 8.
The present invention also relates to nucleic acid sequences encoding an SR-p70 protein or fragments or derivatives thereof biologically active.
More preferably, the invention is directed to a sequence of nucleic acids isolated selected from:
a) the sequence SEQ ID n ~ 1;
b) the sequence SEQ ID No. 3;
c) the sequence SEQ ID n ~ 5;
d) the sequence SEQ ID No. 7;
e) the sequence SEQ ID No. 9;
f) the sequence SEQ ID No. 11;
9) the sequence SEQ ID No. 12;
h) the sequence SEQ ID No. 14;
i) the sequence SEQ ID No. 16;
j) the sequence SEQ ID No. 18;
(k) nucleic acid sequences which are specific for hybridizing specifically to the sequence SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 18 or their sequences do not "Laifes", or to hybridize specifically to their sequences provided;
I) sequences derived from sequences a), b), c), d), e), fl, 9), h), i), j) or k) 25 shows the genetic degeneracy of the genetic code According to a preferred embodiment, the invention relates to the sequences SEQ ID No. 5, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16 and SEQ ID No.
18 co ~ po "a" l respectively to the cDNAs of the human proteins of the sequences SEQ IDN ~ 6, SEQ IDN ~ 13, SEQ IDN ~ 15, SEQ IDN ~ 17 and SEQ IDN ~ 19.
The various sequences which are not known in the invention may be of origin artificial or not. It can be DNA or RNA sequences, obtained by screening sequence banks using probes developed on the basis of sequences SEQ ID No. 1, 3, 5, 7, 9, 11, 12, 14, 16 or t8. Such banks can be prepared by classical ecology of molecular biology, known to those skilled in the art.

.

WO 97/28186 PCT ~ R97 / 00214 -The nucleotide sequences according to the invention can also be prepared by chemical synthesis, or by mixed methods including modification chemical or enzymatic sequence obtained by screening banks.
These nucleotide sequences allow the realization of nucleotide probes able to hybridize strongly and specifically with a nucleic acid sequence keys of genomic DNA or messenger RNA. encoding a polypeptide according to the invention or a biologically active fragment thereof. Such probes make also part of the invention. They can be used as a diagnostic tool in vitro for detection by hybridization experiments. specific transcripts polypeptides of the invention in bi ~ o ~ iq ~ les chords or for the implementation evidence of synthesized drugs or ano, genetic aliases such as the loss of heterozygosity or genetic rearrangement, resulting from a polymorphism, demutations or a different episode.
The probes of the invention are at least 10 nucleotides and at most Co, l, ~ o, len ~ has all the sequence of the ~ ene SR-p70 or its cDNA contained forexample in a cos ", ide.
Among the shortest probes, that is to say about 10 to 20 nucleotides, the conditions of hy ~ iddlion appr ~ u, iées correspond to the stringent conditions usually used by those skilled in the art.
The temperature used is preferably between Tm -5 ~ C at Tm -30 ~ C, preference still between Tm -5 ~ C and Tm -10 ~ C Tm being the melting temperature temperature al ~ que'l ~ 50% of DNA strands appa (ies separate.
Healing is preferably carried out in solutions with high ionic strength.
such as ncl ~ ""., ~ r, l solutions 6 x SSC
In the following manner, the hyaline con¬ tients used are as follows:
- you,.) perdtur ~: 42 ~ C, - ~. ", ~, o., h ~ yllidalion: 6 x SSC, 5 x Denhart's, 0 1% SDS.
as described in Example lll Advantageously, these probes are represented by the following oligonucleotides or their complementaries:
SEQ ID NO: 20: GCG AGC TGC CCT CGG AG
SEQ ID NO: 21: GGT TCT GCA GGT GAC TCA G
SEQ ID NO: 22: GCC ATG CCT GTC TAC AAG
SEQ ID No. 23: AGC ACC TGG TTG ACG GAG
SEQ ID NO: 24: GTC AAC CAG CTG GTG GGC CAG
SEQ ID NO: 25: GTG GAT CTC GGC CTC C

~ PCT FR97 / 00214 .

SEQ ID NO: 26 AGG CCG GCG TGG GGA AG
SEQ ID NO: 27 CTT GGC GAT CTG GCA GTA G
SEQ ID NO: 28: GCG GCC ACG ACC GTG AC
SEQ ID NO: 29 GGC AGC TTG GGT CTC TGG
SEQID # 30: CTG TAC GTC GGT GAC CCC
SEQID # 31: TCA GTG GAT CTC GGC CTC
SEQID # 32: AGM GGA CGC AGC GAA ACC
SEQ ID NO: 33: CCA TCA GCT CCA GGC TCT C
SEQ ID NO: 34: CCA GGA CAG GCG CAG ATG
SEQ ID NO: 35: GAT GAG GTG GCT GGC TGG A
SEQ ID NO: 36: TGG TCA GGT TCT GCA GGT G
SEQ ID NO: 37: CAC CTA CTC CAG GGA TGC
SEQ ID NO: 38: AGG AAA ATA GAA GCG TCA GTC
SEQ ID No. 39: CAG GCC CAC l ~ G CCT GCC
SEQ ID NO: 40: CTG TCC CCA AGC TGA TGA G

Preferably the probes of the invention are marked pre ~ lahlcment to their ulilisdlion. For that reason, more of them are at the door of the man of the trade (radioactive fluorescent labeling chemiluminescent enzymatic etc).
The diagnostic methods in Yifro in which these nucleotide probes are implementations are included in the subject of the present invention.
These methods relate, for example, to the detection of abnormal syntheses (e.g.
accumulation of transcripts) or genetic ano ~ s such as loss of heterozygosity and rea., genetic abnormality and point mutations at Nucleic acid sequence level of nucleic acids encoding a protein SR-p70, according to the above-mentioned data.
The nucleotide sequences of the invention are also useful for the fab (ica ~ ion and the use of oligonucleotide primers for sequencing reactions or a specific method according to the so-called PCR technique or any variant thereof.
(Ligase Chain Reaction (LCR) ..).
Preferred pairs of α,, are chosen from the nucleotide sequences: SEQ ID NO: 1: monkey sequence of 2874 nucleotides and SEQ ID NO: 5: human SR-p70a cDNA "ota""""upstream of the initiation ATG codon and downstream of the translation stop codon TGA.
Advantageously these primers are reprized: senlées by the following couples:
-PCT ~ R97 / 00214 - couple n ~ 1:
meaning primer: GCG AGC TGC CCT CGG AG (SEQ ID NO: 20) antisense primer: GGT TCT GCA GGT GAC TCA G (SEQ ID No. 21) - n ° 2:
primer meaning: GCC ATG CCT GTC TAC AAG (SEQ ID No. 22 ~
antisense primer: ACC AGC TGG TTG ACG GAG (SEQ ID NO: 23) - couple n ~ 3:
meaning primer: GTC AAC CAG CTG GTG GGC CAG (SEQ ID NO: 24) antisense primer: GTG GAT CTC GGC CTC C (SEQ ID No. 25) - couple n ~ 4:
meaning primer: AGG GCG GCG TGG GGA AG (SEQ ID No. 26) antisense primer: CTT GGC GAT GCA CTG GTA G (SEQ ID NO: 27) - couple n ~ 5:
meaning primer: GCG GCC ACG ACC GTG A (SEQ ID NO: 28) primer anlisens: GGC AGC TTG GGT CTC TGG (SEQ tD n ~ 29) - section n ~ 6:
primer meaning: CTG TAC GTC GGT CCC GAC (SEQ ID N ~ 3 ~) a.lllisens primer: TCA GTG GAT CTC GGC CTC (SEQ IDn ~ 31) - step n ~ 7:
meaning: AGG GGA AGC AGC GAA ACC (SEQ ID NO: 32) primer ar ~ lisens: GGC AGC TTG GGT CTC TGG (SEQ ID No. 29) -couplen ~ 8:
meaning primer: CCCCCCCCCCCCCCN (where N is equal to G, A or T) antisense primer: CCA TCA GCT CCA GGC TCT C (SEQ ID NO: 33) - Part n ~ 9:
meaning primer: CCCCCCCCCCCCCCN (where N is equal to G, A or T) ANTISISA: CCA GGA CAG GCG CAG ATG (SEQ ID NO: 34) CA 02245242 l998-07-28 PCTN ~ R97 / 00214 - couple n ~ 10:
meaning primer: CCCCCCCCCCCCCCCN ~ where N equals G, A or T3 antisense primer: GGC GAT CTG GCA GTA G (SEQ ID NO: 27) - Part n ~ 11: *
meaning primer: CAC CTA CTC CAG GGA TGC (SEQ ID NO: 37) antisense primer: AGG AAA GAA ATA GCG TCA GTC ~ SEQIDn ~ 38) - section n ~ 12:
meaning primer: CAG GCC ACC TTG CCT GCC (SEQ ID No. 39) antisense primer: CTG TCC CCA AGC TGA TGA G (SEQ ID NO: 40) These primers correspond to the sequences respectively:
nucleotide n ~ 124 to nucleotide n ~ 140 on SEQ ID n ~ 1 and nucleotide n ~
1 to nucleotide n ~ 17 on SEQ ID n ~ 5 for SEQ ID N ~ 20 nucleotide n ~ 2280 to nucleotide n ~ 2262 on SEQ ID n ~ 1 and nucleotide n ~ 2156 to nurlQotide 2138 on SEQ ID No. 5 for SEQ ID No. 21 nucleotide n ~ 684 to nucleotide n ~ 701 on SEQ ID n ~ 1 for SEQ ID N ~ 22 - nucleotide n ~ 1447 to nur ~ eotide n ~ 143Q on SEQIDn ~ 1 and the nucleotide 1324 to nucleotide 1307 on SEQ ID No. 5 for SEQ ID No. 23 nucleotide 1434 to nucleotide 1454 on SEQ ID No. 1 and nucleotide 1311 at nur ~ eotide1331 on SEQ ID n ~ 5 for SEQ ID n ~ 24 from nurieotide 2066 to nucleotide 2051 on SEQID n ~ 1 and nucleotide 1940 in the nuc, 1925 wind on SEQ ID No. 5 for SEQ ID No. 25.
from nucl ~ 3tide 16 to nuclide olide 32 on SEQ ID n ~ 5 for SEQ ID n ~ 26 from nucleotide 503 to nucleotide 485, SEQ ID No. 5 for SEQ ID No. 27 from nuciéotide 160 to nurle ~ tids 176 on SEQID n ~ 11 for SEQ ID No. 28 - from 1993 to 1976 nucleotide on SEQID n ~ 5 for SEQ ID n ~ 29 nucleotide 263 to nur ~ eQtide 280 on SEQ ID No. 11 for SEQ ID No. 30 from n ~ rleoti ~ the 1943 to nucleotide 1926 on SEQ ID n ~ 5 for SEQ ID n ~ 31 from nucleotide 128 to nur ~ otide 145 on the nucleotide sequence represented in fi ~ ure 22 for SEQ ID No. 32 nucleotide 1167 to nur ~ 1149 olicie on SEQ ID n ~ 5 for SEQ ID n ~ 33 nucleotide 928 to nucleotide 911 on SEQ ID No. 5 for SEQ ID No. 34 from nucioteotide 677 to nucleotide 659 on SEQ ID No. 5 for SEQ ID No. 35 from nucleotide 1605 to nucleotide 1587 on SEQID n ~ 5 for SEQID n ~ 36 CA 02245242 l998-07-28 WO 97/28186 PCT ~ FR97 / 00214 -from nucleotide 1 to nucleotide 18 on the nucleotidic sequence represented at FIG. 13 for SEQ ID No. 37 from nucleotide 833 to nucleotide 813 on the nucleotide sequence represented in FIG. 13 for SEQ ID No. 38 from nucleotide 2 to nucleotide 42 on the nucleotide sequence represented at Figure 13 for SEQ ID No. 39 from nucleotide 506 to nucleotide 488 on the nucleotide sequence represented in FIG. 13 for SEQ ID No. 40 The nucleotide sequences according to the invention may also have uses in gene therapy, in particular for the control of apop-tose and reversion of the transtur ... alion.
The nucleotide sequences according to the invention can moreover be used for the production of recombinant proteins, SR-p70, according to the derivation, which was given at this term.
These protein !; can be produced from defined nucleotide sequences above, according to product production techniques, which are known in the art.
the skilled person. In this case, the sequence nu ~ leolid; used kills is placed under the It gives the signal little or nothing to its expression in a cellular host.
An erricac ~ system for producing a recombinant protein requires have a vector, for example of plasmidic or viral origin, and a cell compatible host The cellular host can be selected from procar ~ rotated systems, such as bacteria ries, or euGaryotes, such as yeasts, cell ~ ~ es insects, CHO
(cell ~ ~ 'es of ovaries of ha ", ~ r Chinese) or any other system advantageously Disponil the ~. A preferred cellular host for the expression of the proteins of the invention is constituted by the bacterium ~ co / i, nota -, -t the strain MC 1061 (Clontec).
The vector must compo, Ler a p, u ,, uhur, signals ~ I initialization and termination of translation, as well as the regions ~ pp, up ~ iées of regulation of the transcription. He must can be stably maintained in the cell and can possibly possess particular signals specirianl the secretion of the translated protein.
These different signals of co-l ~ 'e are chosen according to the cellular host used. AT
this effect, the nucleotide sequences according to the invention can be inserted into vector-replication vectors within the chosen host, or vectors integrative of the chosen host. The vectors will be prepared according to the methods co -., am .., ent used by those skilled in the art, and the resulting clones can be PCTrFR97 / 00214 WO 97/281 ~ 6 introduced in an appropriate host by standard methods, such as for example electroporation.
Expression expression cloning vectors containing at least one of the nucleotide sequences defined above are also part of the present invention.
A preferred cloning and expression vector is the plasmid pSE1 which comprises the the elements necessary for its use as a vector of cloning in E. coli (origin of replication in E. coli and gene resistance to a, l, picillin, from plasmid pTZ 18R), and as an expression vector in animal cells (promoter, intron, polyadenylation site, origin of virus replication) SV40), as well as the elements pe, lllelLanl its single-stranded copy for the purpose of se ~ uençage (origin of the ~; on phage fl).
The characteristics of this plasmid are described in application EP 0 506 574.
Its construction, as well as the integration of cDNAs from acid sequences The nucleic acids of the invention are furthermore described in the examples below. According to a preferred embodiment, the proteins of the invention are in the form of fusion proteins, nolamll, enl as protein fused with glutathione S-transferase tGST). An expression vector designated in this case is represented by the vector plasmidlidi4-1e pGEX-4T-3 (Dl lall.lacia ref-27.4583).
The invention also aims at the host cells expressed by these previous vectors.
These cells can be obtained by intro ~ ction in host cells of a nucleotide sequence inserted into a vector as defined above and the setting in cultivation of cell cultures in conditions that may lead to replication and / or the e ~ cession of the transfected n ~ cti ~ fic sequence.
These cells are useful in a production of a polypeptide Sequence recombinant SEQ ID n ~ 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. ~ 8, SEQ ID No. ~ 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16 or SEQ ID No. 18 or any rle.y "len ~ or drift ~ actively active thereof.
The ~ lllode of pro ~ uction of a polypeptide of the invention in corrolanante form is itself compiled in the present invention, and is acleri ~ e in that one This is the reason for the growth of this type of information in con-jilions allowing the expression of a recombinant polypeptide of SEQID sequence n ~ 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No.
ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16 or SEQ ID No. 18, or any fragment or biologically active derivative thereof, and that we recover ledi ~
polypeptide recom ~ inanl.
The process of filtration used is known to those skilled in the art. The polypeptide recGIll ~.) anl can be purified from lysates and cell extracts, from -supernatant of the culture medium, by methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity techniques using specific mono or polyclonal antibodies, etc.
A variant ~ referred is to produce a recombinant polypeptide) fused to a "Carrier" protein (chimeric protein). The advantage of this system is that it allows a stabilization and a decrease in the proteolysis of the recombinant product, a increase in solubility during in vitro renaturation, or a simplification cation of the purification when the fusion partner has affinity for a specific ligand.
Advantageously, the polypeptides of the invention are fused with glutathione S-transferase in the N-terminal position ("GST" Pharmacia system). The product of fusion is in this case detected and quantified thanks to the enzymatic activity of the GST.
The colorimetric reagent used is a glutathione acceptor, substrate of GST. The recombinant product is purified on a support of chtol "alog, aphie to which were previously coupled glutathione Il ~ es.
Mono or polyclonal labels which recognize a particular SR-p70 protein according to the preceding cleriniLion """ent are also part of the invention. Polyclonal anticG, I, s can be obtained from the serum of a animal immunized against peatine, produced for example by recombina;
Genetics according to the method described above, according to the usual operating modes.
Anlico ".s" ~ onoclG, -a.Jx can be obtained according to the classical method of Hybrid culture is described by Kohler et al., Nature, 1975, 256, 495-497.
Advantageous drugs are antibodies, directed against the central region.
co ", taken between the residue 110 and the residue 310 for the sequences SEQ ID n ~ 2 or 6 or between the residue 6Q and the residue 260 for the sequence SEQ ID No. 8 The "salts" according to the invention are, for example, chimeric, ethyl, ethyl, ethyl, ethyl, and F (ab ') 2 derivatives. . They can also present as immunoconjugates or labeled anilico, ps.
Furthermore, they may be used for the purpose of promoting recombinant Ls polypeptides.
the antigens of the invention, in particular the monoclonal alcohols, can also be used for the detection of these polypeptides in a sample organic.
They thus constitute a means of analysis immunocytochirl ~;
Immunohistochemistry of the ex ~ n: ssior) of SR-p70 proteins on tissue sections WO 97/28186 PCTA ~ R97 / 00214 specific, for example by immunofluorescence, gold marking, enzymatic immunoconjugates.
In particular, they make it possible to demonstrate an accumulation of SR-p70 proteins in certain tissues or biologic preparations, which makes them 5 useful for the detection of cancers or monitoring the evolution or remission of preexisting cancers.
More generally, the antibodies of the invention can advantageously be in any situation where the expression of an SR-p70 protein must be observed.
The invention therefore also relates to an in vitro diagnostic method for pathologies correlated with ano-lll expression or accumulation of SR-p70 proteins, in particular, the phenomena of co-ionization, from a biogenic sampling, characterized in that at least one of the compounds of the invention is brought into contact with said biological sample, under conditions permitting the eventual alteration specific immunological complexes between an SR-p70 protein and the at least one antibodies and in that the CGIIl, the specific immunological lesions, are detected.
formed.
The invention also conceives an <it for the ciiagnoslic in vitr ~ of an expression or an accumulation of SR-p70 proteins in a biological sample and / or for the measurement of the rate of e ~ ,, essiol- thereof in said sample comprises ing:
- at least one ~ nlicol ~ Js speciti ~ e a protein SR-p70, possibly fixed on a support, - Revealing means of r ~ "alion of anti ~ enen / antibody complexes specimens between an SR-p70 protein and said antigen and / or the union of these complexes.
The invention also relates to a method of early dia ~ noslic training destumers, by the demonstration in the serum of an individual, auto-anlicor ~ s directed against an SR-p70 protein.
Such a method of early diagnosis is characterized in that contact a serum sample taken from an individual with a polypeptide of the invention, possibly fixed on a support, under conditions pe, ellanl the fo ", ~ alio" of co, r, A: q ~
auto-anoleol ~ .s possibly present in the serum), and in that one detects the specific immunological events that may be formed.
The invention also relates to a method for determining a variety of allelic, mutation, deletion, insertion, loss of heterozytosis , WO 97/28186 PCT ~ R97 / 00214 -or a genetic abnormality of the SR-p70 gene. may be involved in characterized in that it uses at least one nucleotide sequence described above. Methods of Determining Allelic Variability of a mutation of a deletion of an insertion of a loss of heterozygosity or a genetic anomaly of the SR-p70 gene we prefer the method ca ~ d- lerlsed in this that it includes at least one step of ~ ", pliricclion by PCR of the sequence target nucleic acid of SR-p70 susceptible to polymorphism, mutation, deletion, or insertion using a pair of sequence primers nucleotides defined above a step in which we proceed to treatment of the products of pliriés using resl enzyme, i ~ lio, ap ~, oprié and a step in which the detection or the determination of at least one products of the enzymatic reaction.
The invention also discloses pharmaceutical compositions which are as active ingredient a polypeplide, answering the preceding definitions preferentially in the form of its light 5550cie to a phased vehicle "" aceutically ~ ~ Hle CCPS.
Such con posilions offer a new approach to deal with the phenomena of cancer at the level of the cornea, the degree of multirling and the dirterencialior, cellular.
Preferentially these co ", po ~ ilions can be Eid", n; ~ L, ees systemically preferably intravenously intradermally or intramuscularly oral route.
Their modes of adm ~ (cilion dosages and galenic forrres optimal can be determined in accordance with the general criteria and taken into account in the elaboration of l ~ dile ", therapeutic treatment adapted to a patient such as age or weight cGr, uGret of the paffent the gravit ~ of its state gené ~ al tolerance to the treatment and the secondary effects, conslds, etc.
Finally, the present invention provides a method for gene therapy in which the immunizations which are coded for an SR-p70 protein are 1 year old at the same time. Cibies via inactivated viral vectors.

Other features, advantages and disadvantages of the invention appear later in this document.
description with examples and figures whose legends are shown below.
after.

~ PCT FR97 / 00214 ? EGENDE OF FIGURES

Figure 1: Nucleic comparison of the monkey SR-p70a cDNA (corresponding SEQ ID NO: 1) with the nucleic sequence of the monkey p53 cDNA.

Figure 2: Protein comparison of monkey SR-p70a with the p53 protein of monkey (sw: p53-cerae).

Figure 3: Comparison of the nucleic acid sequence of SR-p70a cDNA and b monkey (corresponding respectively ~ SEQ ID No. 1 and SEQ ID No. 3).

Figure 4: Nucleic Sequence and Protein Sequence Derived from SR-p70a from monkey.
Figure 5: Partial nucleic sequence and complete deduced protein sequence of SR-p70b of monkey.

Figure 6: Partial nucleic sequence and complete protein sequence deduced of SR-p70a human (cGr, ~ sponding to SEQ ID No. 5).

Figure 7: Partial nucleic sequence and complete deduced protein sequence of Mouse SR-p70c (cGI1 ~ spondanl to SEQ ID No. 7).

Figure 8: Sequence nuc! E; q-the pa, I; ~ lle and deduced protein sequence partial of SR-p70a mouse (horn, ~ ponddnl SEQ ID No. 9).
Figure 9: Multi-alignment of proteins deduced from monkey SR-p70 cDNAs (a and b) human (a) and mouse (a and c).

Figure 10a: Immune Immunoblot of SR-p70.

Figure 1Qb: Detecliei) of the SR-p70 endogenous protein.

WO 97/28186 PCT ~ FR97 / 00214 -Figure 11: Chromosomal location of the human SR-p70 gene. The signal appears on chromosome 1 in the p36 region.
,.
Figure 12: Genomic structure of the SR-p70 gene and comparison with that of the d. p53 gene. The human protein sequences of SR-p70a ( top of the alignment) and the p53 (bottom line) are broken up into peptides according to the respective exons from which they are codes. The numbers at the arrow level correspond to the numbering of the corresponding exons.

Figure 13: Human genome sequence of SR-p70 from 3 'of intron 1 until 5 'of exon 3. The introns are framed. At positions 123 and 133 are localized two variable nucleotide positions (G ~ A in 123 and C ~ T in 133). The sites of ~ esl ~ i ~ lion of the enzyme Styl-are underlined (position 130 in the case where there is a T instead of a C at position 133 position 542 and position 610). The arrows posiersnent the ", Nucleus gr es used in the example X ~.

Figure 14: (5 'nucleotide junction of human cDNAs of SR-p70d and SR-P70a.

Figure 15: Mullial, ~ "e", ent nu ~ l ~ sequences corresponding to the SR-p70 human abde etf.

Figure 16: ~ 1ulti al: g ~ ei ~ en ~ proteins of ~ uites human SR-p70 cDNAs (ab ~ d eetf).
Figure 17: Naked sequence: Partial eiql: e and partial deduced protein sequence of the SR-p70a human. The two bases in ca, a ~ lères gras correspond to two variable positions (see Figure 6). This sequence presents a 5 'non-coding region more con ~! ete than that shown in the figure 6.
Figure 18: Analysis of transcripts SR-p70a after the plif; a ~ ion by PCR.
track M: weight markers, ~ 'éleul ~ ires "1 I <b ladder "(GIBCO-BRL) WO 97/28186 PCT ~ R97 / 00214 lane 1: line HT29 lane 3: SK-N-AS line lane 5: UMR-32 line lane 7: U-373 MG line lane 9: SW 48Q line lane 11: CHP 212 line lane 13: SK-N-MC line tracks 2 4 6 8 10 12 14: negative indicators corresponding to the tracks 101 3 ~ 7 9 11 and 13 respectively (absence of transc, i, reverse utase in the RT-PCR reaction).

Figure 19: A: Analysis by Agarose Gel Electrophoresis of the Fragments amplified by PCR (from the 3 'of the intron 1 ius ~ u ~ to 155 'of exon 3). The number, ota ~ ion tracks cor, e ~ spond to the numbering of the control sample. Track M: markers of molecular weight ~ es ("1 kb ladder").
B: Analysis identical to that of the party ~ A after a digestion by the enzyme of resl, i ~ liori styl of the same ecl ~ anlillons.

Figure 20: Repression "schematic map with a map of reaL, i ~ lion partial of pCDNA3 containing human SR-p70a.

WO 97/28186 PCT ~ FR97 / 00214 EXAMPLE I

Cloning of SR-p70 cDNA from COS-3 cells.

1 Celtule culture COS-3 COS-3 cells (African green monkey kidney cells transformed by SV 40 virus T antigen) are cultured in DMEM medium (GIBCO-BRL ref. 41 964; 047) containing 2 mM L-glutamine and supplemented with 50 mg / l of gentamycin and 5% fetal bovine serum (GIE3CO-BRL reference 10231-074) until semi-confluence.

2. Preparation of the messenger RNA
a) extraction of the mess ~ ger RNA
The cells are recovered as follows:
the adherent cells are washed twice with ta, ~ "~ on PBS (phosphate buffered saline reference 04104040-GIBCO-BRL) then scraped with a scraper rubber and centrifuged.
The clotular pellet is suspended in the composition Iyse's tdlllpon following: guanidine-thiocyanate 4M; 25mM sodium citrate pH 7; sarcosyl 0.5 %; 0.1 M. The suspension is sonicated using a sonicator Ultra-Turrax n ~ 231256 (Janke and Kundel) at maximum power ", ale pendan ~ a minute. Sodium acetate pH 4 is added up to 0 2 M. The solution is extracted with a volume of a mixture phenol / cl, lo, or ~ "", e (v / v; 5/1). We precipitate at -2Q ~ C the RNA contained in the a ~ uellse phase using a volume of isopropanol.Le pellet is resuspendu in the ld, npon lysis. The solution is again extracted with a mixture phenol / cl, lorofo ", 1e and the RNA is precipitated with isopropanol.
After washing the pellet with 70% ethanol then 100% the RNA is resuspendu in water.
b) PuriricaliG "of r, a ~ .liol1 poly A ~ RNA
The purifical: ion of the poly A ~ fraction of the RNA is produced using the Dynabeads kit oligo (dT) 2 ~ of DYNAL (reference 610.05) according to p, ~ Jlocole recommended by the The principle is based on the use of polystyrene beads super-pardr "aynetique on which is attached a oligonucleotide poly (dT) 25. The fraction poly A ~ of the RNA is hybridized on the oligo (dT) 2s coupled to the beads that we trap on a magnetic medium.

WO 97/28186 PCT ~ R97 / 00214

3. Constitution of the complementary DNA bank a) preparation of the complementary DNA
From 0.5 ~ 9 of the poly-A RNAs of COS-3 cells obtained at the end of the step 2, the 32 P.dCTP-labeled single-stranded DNA (DNA) was prepared supplement obtained has a specific activity of 3000 dpmJng) with r the following sequence synthetic primer (comprising an E3amHI site):
5'CGATCCGGGCC CIII m IIIIT 1 1 <3 ' in a volume of 30 ~ I of composition buffer: Tris I ICI 50 mM pH 8.3, 6 mM MgCl 2, 10 mM Drr, 40 mM KCl, containing 0.5 mM each of deoxynucleotide triphosphates, 30 ~ Ci of dCTP a32P and 30 U of RNasin (prome-ga). After one hour of incubation at 37 ~ C, then 10 minutes at ~ 0 ~ C, then again 10 minutes at 37 ~ C, with 200 units of the enzyme reverse transcriptase RNase H (GIBCO-BRL reference 8064A), 4 μl of EDTA is added.
b) Alkaline hydrolysis of the RNA template 1 ~ 6 ~ I of a solution of 2N NaOH is added, then incubated for 5 minutes at 6 ~~ C.
c) P ~, ~ irionation on ~ nne Sephacryl S400 In order to eliminate the synergetic primer, the cG-1 ~ en ~ enamel DNA is purified on a 1 ml column of sephacryl S400 (ν max), equilibrated in TE buffer.
The first two radioactive waves are grouped and precirculated with 1/10 volume of a solution of ammonium acetate 10 M and 2.5 volumes ethanol, this after an e ~ lla- ~ ion, with a volume of chlo, ufo ,, lle.
d) Addition holl, opolymer ~ ue dG
The 3 'DNA is extended with a dG "tail" with 20 units of the enzyme "aalte, ansrerase" (Pharmacia 27073001). I of Idl ~ pon co-loci ~ ion: Tris HCl 30 mM pH 716; cobalt chloride 1mM, acid 140 mM cacodylic acid, Drr -1, 1 mM, 1 mM dGTP, for 15 minutes at 37.degree.
2 ~ I of 0.5 M EDTA is added ~ 3) Repeat steps b) e ~ c) f) Pairing of cloning vector pSE1 (EP 506,574) and DNA
complementary in the presence of the adapter.
The pellet is centrifuged and dissolved in 33 μl of TE buffer.
ng) of clona5 ~ e pSE1 vector, 1 ~ 1 (120 ng) of the following sequence adaplalene vante (including a Apal site):
5'AAAAAAAAAAAAAGGGCCCG3.

PCTrFR97 / 00214 10 ~ 11 of a solution of 200 mM NaCl, incubated for 5 minutes at 65 ~ C then the reaction mixture is allowed to cool to room temperature.
g) Ligation The cloning vector and single-stranded cDNA are pooled in a volume of 100 μl with 32 units of phage T4 DNA ligase enzyme (Pharmacia Reference 270 87002) overnight at 15 ~ C in a composition buffer: Tris HCI 50 mM p ~ 7.5, 10 mM MgCl 2, 1 mM ATP.
h) Synthesis of the second strand of the cDNA
We eliminate! phenol extraction followed by extraction with ~ illor ~ fo "" e ~, then add 1 / 10th of a volume of a solution of ammonia acetate 10 mM, then 2.5 volumes of ethanol. Centrifug, the pellet is dissolved in the composition buffer Tris acetate ~ 3 mM pH 7.9 potassium acetate 62.5 mM, 1 mM magnesium acetate and 1 mM dithiothreitol (Drr), the second strand of DNA
complementary is syn1: hetized in a volume of 30 ~ 1 with 30 units of the enzyme Phage T4 DNA polymerase (Pha-nacia, reference 270718) and a mixture of 1 mM of the four deoxynucleotide triphosphates of dATP, dCTP, dGTP and dTTP, as well as two units of the phage T4 gene 32 protein (Pha ~ n, acia, reference 27-0213)) for one hour at 37 ~ C is extracted with phenol and removed traces of phenol by a P10 polyacrylamide column (Biogel P10-200-400 mesh -, 15011050 - Biorad).
i) transro "" dlion parelec; t, opordlion E. coli MC 1061 cells are transformed with the recombinant DNA obtained preceded "~." el) l by electropol ~ ion using the device Biorad Gene Pulser (Biorad) used at 2 ~ kV in co-dilutions prescribed by the fabncan ~ then grow the bacterium ~) danl one hour in medium said middle L3 (Sambrook op cit ~) of co ~ osilio,): bactotryptone 1Q g / l; yeast extract 5 g / l; NaCl 10 g / l.
The number of independent clones is determined by a dilution at 1 / 1000th of l.ansrur ,,, alion after the first hour of incubation on a box LE ~ medium supplemented with 1.5% agar (w / v) and 100 ~ g / ml ampicillin, called subsequently the medium LB agar. The number of independent clones is 1 million.
J) Analysis of the cDNAs of the bank In the context of the analysis of individual clones of the bank by a Nucleic acid sequencing of the 5 'cDNAs, a clone, called SF'<- p70a, was revealed to present a hGIl ~ ~ o ~; c pa, ~; el '~ with the cDNA of the already known protein, the p53 protein (Genbank X 02469 and X 16384) (Figure 1). The sequences were with the kit United States Bio ~ he, r .: ~~ '(reference 70770) and / or the kit Applied .
Biosystems (references 401434 eVou 401628) using the method of Sanger et al. Proc. Natl. Acad. Sci. USA; 1977 14 5463-5467. Plzsmidic DNA is prepared from the kit WIZARD mini preparation (Promega reference A7510). The The primers used are 16 to 22 mer oligonucleotides complementary to either S to the vector pSE1 in the region immediately 5 'of the cDNA is to the sequence cDNA.
A second cDNA was isolated from the same bank by screening similar to the technique described in EXAMPLE III (3) below with a fragment of AI:) N SR-p70a marks at 32p with the RRL kit "Random Primers DI ~ IA labeling"
systems '' (reference 18187-013). Hybridization and iava ~ e buffers are added with 50% of fo, ma "" ~ e. The last wash is done in 0 1 x SSC / SDS
0 1% to 60 ~ C This second sequence (cDNA SR-p70b) is identical to the first but has an internal fragment deleted (Figure 3).
The two SR-p70 cDNAs with a length of 2874 nucleotides (SR-p70a) and 2780 1 ~ nucleotides ~ SR-p70b) cs "respond to the products of a single gene splicing alternatively, deleting a 94 base deletion between nucleotides 1637 and 1732 and a premature termination of the coded co.processed protein.
deduced from the two cDNAs respectively 637 amino acids and 499 amino acids (Figures 4 and 5).

Ol ~ tenlion of the sequence and cloning of the cDNA of the protein SR-p70a from HT-29 cells (adenocarcinoma, human colon).

1 ~ Cu / tur ~ cell ~ 3s HT-29 C ~ Hu'es are grown in McCoy medium (GIBCO 26600-023) supplemented with 10%
of fetal calf serum (GIBCO 10081-23) and 50 mg / l of gentamycin to semi-3 ~ confluence 2) Preparation of the complementary DNA
Messenger RNA is prepa, ~ as described in EXAMPLE 1.2. The cDNA is prepared so, for example, that described in EXAMPLE 1.3 with 5 μg of messenger RNA
3 ~ total using an a ", we have polytT) t2 The .eaclion is not interruptedQ with EDTA.

WO 97tZ8186 PCT / FR97 / 00214 3) Specific amplification of human DNA by PCR technique The polymerization is carried out with 4 ~ I of cDNA in 50 ~ I final with the buffer of following composition: 10 mM Tris I ICI pH 8.3, 2.5 mM MgCl 2, 50 mM KCl presence of 1% DMSO, 0.5 mM dNTP, 4 μg / ml of each of the two primers.
nucleic acid and 2.5 units of Taq DNA polymerase (Boehringer). Couples of primers were selected on the nucleic sequence of the SR-p70 clone of COS-3, especially upstream of the ATG of initiation and downstream of the translation stop ~ GA and are of the following compositions:

meaning: ACT GGT ACC GCG AGC TGC CCT CGG AG
Kpn I restriction site antisense antigen: GAC TCT AGA GGT TCT GCA GGT GAC TCA G.
site of r ~, l, i, lion Xba I

The reaction is carried out for 30 cycles 94 ~ C / 1 minute, 54-60 ~ CI1 minute 3u seconds and 72 ~ C / 1 minute 30 seconds, followed by a last cycle of 72 ~ C / 6 minutes.

4) Obtaining the sequence of the hvmain cDNA
In a first step, the PCR product is removed from the oligonucleotides on a column of sephacryl S400 then dess ~ 'by cb, o "~ aLoy-aphie exclusion on a co-P10 polyacrylamide column (Biorad reference 1504144). The reactions of Sequencing is performed using the Applied Biosystems Kit (reference 41628) with specific cDNA oligonucleotides. The sequence obtained is very similar to that of the monkey SR-p70a and the deduced p, o ~ ei ~ e contains 636 amino acids (Figure 6).
Similarly, other sequences derived from human lines or tissues have were obtained for the codanic part of human SR-p70, no.
Lungs or pancreas The proleins ded ~ litPs of these sequences are identical to those obtained for the line HT-2 ~.

5) Cloning of ~ "human cDNA in plas" L_ ~ pCDNA3 (Inv ~ trogen V 790-20) The PCR product obtained in 3) and the plasmid are digested by the two enzymes 35 de.resllicLio ~ Kpn I and Xba I then purified after migralic ~ n on a 1% agarose gel to Using the Geneclcan kit (Bio 101 ~ efert l) 3105). After ligation with 100 ng of insert WO 97128186 PCT ~ FR97 / 00214 -and 10 ng of vector and transformation (technique described in I ~ ExEMpLE 1.3.g and i Recombinant clones are verified by sequencing using the Applied Biosystems kit citb above.

EXEMPI E lll Cloning of mouse SR-p70 DNA from AtT-20 cells (tumor pituitary) 1J Cu / tur ~ cellulair ~ lis ~ born AtT-20 The cells are cultured in medium Ham F10 (GIBCO 31550-023) supplemented with 15 ~ / 0 horse serum (GIBCO 26050-047) and 25% fetal calf serum (GIBCO 10081-073) and 50 mg / l of gen ~, ycine until serni-confluence.

2) Preparation of the co-ordinate DNA library The library is performed as described in EXAMPLE 1 (2) and (3) from the cells grown above.
3) Cnbtage of the bank a) Preparation of "e", brc., - es The clones of the bank are spread on medium LB agar (petri dishes diameter 150) coated with Biodyne A (PALL, BNNG 132).
37 ~ C the clones are l, c ~ er ~ s by contact on new membranes. These The last ones are treated in deposits ~ nl on Whatman paper 3 mm soaked with following solutions: NaOH 5 N NaCl 1.5 M for 5 minutes then Tris HCl 0.5 MpH 8 1.5 M NaCl, 5 minutes. After treatment with p, oleinase K in the Lampon next: Tris HCl 10 mM pH 8 EDTA 10 mM NaCl 50 mM SDS 0.1%
Proteinase K 100 μg / ml for one hour at the "per lure a", ~ ia te les me ", b, dnes are washed with ~ ~ ondal "" ~ in 2 x SSC (sodium citrate NaCl) dried then incubated in a vacuum oven at 80 ~ C pen ~ ian ~ 20 minutes.
b) Prepa, ~ lion of the probe On the basis of sequences of the monkey and human SR-p70 cDNAs a first sequence was performed on a r ~. ~ ylllelll amplified from the mRNA of line AtT-As described in EXAMPLE 11.3 and 4 with the oligomers of compositions following:

WO 97/2818

6 23 meaning primer: GCC ATG CCT GTC TAC AAG
antisense primer: AGC ACC TGG TTG ACG GAG.
Based on this sequence a mouse specific oligomeric probe was chosen and has the following composition:
CAT G1-G ACC GAC ATT G GAG.
100 ng of the probe are labeled in 3 'with 10 units of Terminal Transferase (Pharmacia) and 100 ~ Ci of dCTP a32P 3000 Ci / mmole (Amersham reference PB
10205) in 10 ~ I of the following buffer: Tris HCI 30 mM pH 7.6 cacodylic acid 140 mM CoC12 1 mM DTT 0.1 mM for 15 minutes at 37 ~ C. Nucleotides Unincorporated radiolabeled compounds are removed on a P10 polyacrylamide column.
(Biorad reference 1504144). The resulting probe has a specific activity of approximately 5.1o8 dpml, ug.
c) Prehybridization and Hybridization Membranes prepared in a) are prehybridized for 30 minutes at 42 ° C in 6x SSC.
15 5 x Denhart's; 01% SDS then hybridized a few hours in the same buffer addition of the probe prepared in b) at a rate of 106 dpm / ml.
(d) Washing and Extinguishing Menl ~ rdlles are washed twice at the ambient level in the 2 xSSC / SDS 0.1% and then one hour at 56 ~ C in 6 x SSC / SOS 0.1%. Hybrid clones 20 are shown with KODAK XOMAT films. A positive clone containing SR-p70 from The mouse is selected here and hereinafter SR-p70c.

4) Sequencing the mouse SR-p70 and analyzing the sequence The sequence is obtained using the Applied Biosystem kit (reference 401628). The pro ~ ei ~ ue sequence deduced from the mouse SR-p70c cDNA (FIG.
very strong with those of the human and monkey except in the N-section.
which is highly divergent (see Figure 9). With the help of tec ~ lni, Ie said PCR
in a manner similar to that described in EXAMPLE 11.3 and 4: a second sequence 5 '(from the same AtT-20 library) was obtained (Figure 8). The sequence N-terminally derived protein (denoted by sequence SR-p70a) is very similar to that deduced from human SR-p70 and monkey cDNAs ~ SR-p70a) (Figure ~). The AtT-20 lineage therefore has at least two SR-p70 transcripts. These last two diverge in the N-terminal part by different splicing.

,.

WO 97128186 PCT ~ R97 / 00214 EXAMPLE IV

1) Product ~ on of SR-p70 recombinant proton in E. coli a) Construction of the expression plasmid It consists in putting the terminal -COOH part of the monkey protein SR-p70a.
from valine at position 427 to histidine -COOH terminal at position 637 in fusion with glutathione Sl ~ ans ~ eiase (GST) plasmid vector pGEX-4T-3 (Pharmacia Reference 27-4583). For this the corresponding insert of the SR-p70a (position 1434 to 2Q66) was amplified by PCR with 10 ng of plasmid containing The monkey SR-p70a cDNA. Naked primers are of the following composition:

primer meaning: m GGA TCC GTC AAC CAG CTG GTG GGC CAG
BamHI restnction site antisense primer: AAA GTC GAC GTC GAT CTC GGC CTC C
Sal I site The fragment obtained as well as the vector are digested by the restriction enzymes Bamtll and Sal I and cloning is carried out as described in EXAMPLE 11.5. The clone selected is called pG SR-p70.
b) Expression and p ~ "iricaliGi ~ of the pn.tei., e fusion GST-pSR-p70 This step was, ~ r ~ - ~ e using the kit "bulk GST p- ~ nficdlion module" (Fl, a "" acia References 27-4570-01).
In this case, the clone ~ ecorr, '~ nanL was cultured at 37 ~ C in a liter of medium 2x YTA + ampicillin 100 ~ lglml. At DO 0 8 the expression is induced with 0 5 mM lPTG for 2 hours at 37 ~ C After centrifugation the cell pellet is taken up in cold PBS then ultrasonically sonicated. After addition of 1% triton X-100 ml are incubated for 30 minutes with stirring at room temperature.
centrifugation at 12000 g for 10 minutes at 4 ~ C the supernatant is recovered. Purification 3 ~ tion is then performed on a column of ch, o ", alog, dphie affinity glutathionsepl ~ a, use 4B.The fixation and washing are ré ~ iisé Id", pon PBS and elution is performed by competitlon with reduced glutathione The final concentration is brought ~ 300 ~ 1g / ml of fusion protein.

PCTA ~ R97 / 00214 -2) Production of SR-p70a protein in COS-3 cells.
The COS-3 cells are transfected with plasmidic DNA pSE1 in which cloned the monkey SR-p70a cDNA (EXAMPLE 1.1) or with plasmid DNA
of the pSE1 vector in tan ~ that control by the DEAE Dextran technique: the cells COS-3 are seeded at 5 x 10 ~ cells per 6 cm dish in culture medium containing 5% fetal bovine serum (EXAMPLE 1.1). After culturing, the cells are rinsed with PBS. 1 ml of the following mixture is added.
1 ~ 9 DNA 250 ~ g / ml DEAE Dextran and 100 ~ M chloroquine. The cells are incubated at 37 ~ C in 5% CO2 for 4 to 5 hours. The medium is aspirated add 2 ml of PBS adds 10% DMSO and the cells are incubated for one minute by stirring the boxes. The medium is again aspirated and the cells are rinsed twice with PBS. The cells are then incubated at 37 ~ C with medium containing 2% fetal bovine serum for the duration of the expression that is usually 3 days old.
The SR-p70a protein is then analyzed as described in EXAMPLE VI by immuno ", p, ~ i, la.

EXAMPLE V

Preparation of specific drugs 150 1l9 of proteins of ~ chanlillon p ~ pa, e according to EXAMPLE IV were used to immunize a rabbit (male to about 1 to 2 kg New-Zealand). The immunizations was done every 15 days according to the pru ~ ocola described by V ~ itl ~ 4 ~ itis Methods in Enzymology, 1981 73 4 ~ i. For the first time, a volume of anti-aging solution It is emulsified by a volume of cGn ~ 'and Freund's adjuvant (Sigma reference4258 ~ Five reminders were administered by adiuvant inco ", plet of Freund (Sigma reference ~ 5CI6).
EXEMPI E Vl Detection of SR-p70 protein "Westem immu"t'llll, lg "(immunoblot) 1 ~ Materials u ~ ilized forl'immmunoe "" ~ r ~ inte a) Cell lines used for the immunoer, ~, einle.
The following cell lines were grown as described in the catalog ~ catalog of cell lines and hyL, ndo ", as 7th edition 19g2> ~ ATCC (American Type CA 02245242 l998-07-28 Culture Collection): COS-3 CV-1 (monkey kidney cell line) HT-29 U-373MG (human glioblastoma), MCF7 (human mammary adenocarcinoma) SKNAS
(human neuroblastoma grown under the same conditions as COS-3) SK-N-MC
(human neuroblastoma) IMR-32 (human neuroblastoma) CHP212 (neuroblastoma 5 human grown under the same conditions as CV-1) Saos-2 (osteosarcoma) SK-OV-3 (ovarian adenocarcinoma) and SW 480 (human colon adenocarcinoma).
b) COS-3 cells transfected with the SR-p70a cDNA.
COS-3 cells were transfected as described in EXAMPLE IV.2. As Control cells were transfected with plastid DNA, which does not contain recombinant SR-p70a cDNA.

2) Pn ~ paration of protein samples from eukaryotic cell culture o ~ J
of cells transfected ~ es.
After culturing, the cells are washed with PBS and then taken up in a La, pon RIPA (PBS with 1% NP40 0 5% sodium deoxycholal, at Q 5% SDS) supplemented with 10 μg / ml RNAse A 20 ~ Lg / ml DNAse 1 2, ug / ml aprolil) and 5 μg / ml leupepli 0 7 ~ lg / ml pepaLdli "e and 170, ug / mt PMSF.The c ~ es are ultrasonically sonicated to 4 ~ C and left for 30 minutes at 4 ~ C. After "~; c, ucenl, ir.lgation to 12 000 rpm is recupère the supernatant. The concentration of prolein is measured by the method of Gladrord.

3) "Westem ~ lolki" ~ "
S or 50 ~ 9 protein (50 ug for cell lines and 5 ug for cells transfected) are put in 0 2 volume of the "eon ~ ~ .l, opl, orese 6X following: Tris HCl 0 35 mM pH 6 8 10 3% SDS 36% glycerol 0 6 mM DTT 0 012% blue from The samples are deposited and migrated in an SDS-Page gel.
10% (30/0 8 Bis) then éle ~ l, ul, ansrérés on a me ", bra-1e nitrocellulose.

4) Revealing by antibodies The residue is incubated for 30 minutes in the TBST blocking agent (Tris HCl).
mM pH 8 NaCl 150 mM 0 2% Tween 20) addilis ~, u ~ é 5% milk (GIBCO-SKIM
MILK) at temperature a "bianle." The ", e", l, r. ~ Is not necessarily brought together of the anlico, ps anti-SR-p70 (aSR-p70) in the same ta, npon 16 hours at 4 ~ C washed 3 10 minutes with TBST, then incubated for one hour at 37 ~ C with second "lico" ~ anti-imm ~ ", rabbit immunoglobulin coupled with peroxidase (SIGMA A055). After three lava ~ es of 15 minutes the revelation is carried out using ECL kit (A ", elaha, t, RPN2106) by chemiluminescence.

WO 97/28186 PCT ~ FR97tOO214 At the same time, the same samples were revealed by an antico ~ us anti-pS3 (a p53) (sigma E3P5312) followed by a second anti-mouse immunoglobulin antibody.

5) Figl ~ res and results.
Example 10 Immunoblotting of the SR-p70 Protein Example 10a: Detection of recombinant SR-p70 protein columns 1 and 3: COS-3 transfected with the vector pSE1.
columns 2 and 4: COS-3 L, dsfe..laed with the plasmid pSE1 conLt: na-l the cDNA of SR-P70a.
columns 1 and 2: Revelation by anilico, ys anti-SR-p70 (aSR-p70).
columns 3 and 4: Revelation with anti-P53 antibody (ap53).
Fiaure 10b: Endogenous Pole Oleinion SR-p70 -columns1: COS-3; 2: CV-1; 3: HT-29; 4: U-373MG; 5: MCF7; 6: SKNAS; 7 SK-N-MC; 8: 1MR-32; 9: CHP212; 10: Saos-2; 11: SK-OV-3et12: SW480.
A: Revelation by the anlicor ~ us aSR-p70.
B: Reveiation by a ~, liCG- ~ 5 ap53.

The a ~ tic ~, ~ s ~ I: ~ R-p70 specifically recognizes the recombinant proteins (Figure 10a) and endo ~ enes (Figure 10b) and does not cross with p53. Lines analysis human cell or monkey shows that the pru ~ eine SR-p70 as the p53 is usually weakly det ~ ~ hl ~. However, when an accumulation of p53 exists the SR-p70 devienl: it also more easily detect ~ hle (Figure 10b). A
RT-PCR study of the distribution of lldlls ~. SR-p70 shows that the gene is expresses in all cell types tested.

EXAMPLE Vll Cloning of the SR-p70 gene and lo ~ -lisal-Qn cl ~, u ,,, osomic.

1) Cloning of the SR-p70 gene The library used is a library of cells prepared with genomic DNA.
Purified Human Placement of La and cG "" made by Stratagene (ref 95 1202).
Cril ~ lage of the gene is réa ~ isé as described in Example 111.3 with a fragment of 32p-labeled SR-p70 DNA with BRL kit "Random Primers DNA Labeling Systems "(reference 18187-013) The hybridization and washing buffers are added with 50% formamide. The last wash is done in 01 x SSCISDS
0 1% at 60 ~ C. In a similar way the SR-p70 gene was isolated from a bank prepared with genomic DNA of the black C57 mouse.

Analysis and partial sequencing of the clones highlight the presence of 14 exons with a structure close to that of the gene p53 notal ~ "" ent in the part central or the size and positioning of the exons are very conservative (Figure 12).
This structure has been partially defined in mice and humans By way of example ~ the human genomic sequences of the 3 'of the intron 1 of the exon 2 of intron 2 and of exon 3 are shown in FIG.

2) Chromosomal oc ~ lisation of the SP ~ -p70 gene in humans It was performed with human SR-70 gene DNA using teclene described by R. Slim et al. Hmm. Broom. 1991 88 21-26. Fifty mitoses were analyzed, of which more than 80% had double spots located in 1 p3B on both ~ I "or" oso ", es and more particularly in 1p36.2 -1p36.3 (Figure 11) The idenl, ficdlion chromosome 1 and its orientation are b ~ sées on the heterocl ~ ro "aline of the col, sl, iclion seconda; r ~. The images were made on a Zeiss Axiophot microscope captured by a LHESA chilled CCD, and processed by Optilab.

EXAMPLE Vlll A) Introduction of mRNA coding for a deduced human SR-p7α protein presenting at the same time a shorter N-terminal e ~ mite and a divergence.

1) Cultures of cPl ~ Jes IMR-32 fneuruh, 'ah ~ mQ hvmafn) The cells were cultured as described in the catalog "catalog of cell lines"
and hy ~, ido ") as, 7th edition 1992" of the ATCC ~ American Type Culture Colection).
2) Preparing the AONc RNA is phephrb as described in Example 1.2.a. The cDNA is pr ~: ~ ua, ~ so similar to that described in Example 1.3 with ~ ug of total RNA in a final volume 20 μl using a poly (T) 12 primer and with cold nucleotides. The reaction is not interrupted with EDTA.

, 3) Specific amplification of the AONc SR-P70 by the so-called PC tecnnic Polymerization is carried out with 2 μl of cDNA in 50 μl final with the buffer of following composition: 50 mM Tris HCl pH 9 2 16 mM (NH4) 2 SO4 1 75 mM MgCl2 in the presence of DMSO 10% NTP 0.4 mM 100 ng each nucleic primers and 35 units of the mixture of Taq and PWO polymerases (BoehringerMannheim Ref 1 ~ 81 842).
The pair of trees is of the following size:
meaning primer: AGGCCGGCGTGGGGAAG (position 16 to 32 Figure 6) antisense primer: CTTGGCGATCTGGCAGTAG (position 503-485 Figure 6).

The reaction is carried out for 30 cycles at 95 ~ C / 30 seconds 58 ~ C / 1 minute and 68 ~ C / 2 minutes 30 seconds followed by a last cycle 68 ~ C / 10 minutes.
The product PCIR is subjected to an elecl, ophG, t: se. on a 1% ~ buffer agarose gel T ~ E). After staining with ethidium bromide two major bands are recovered: a band of about 490 bp in size (expected size (see Figure 63) and one band additional approximately 700 bp in size. The latter is extracted from the gel using of the kit "geneclean" (Bio 101 ref 1001 400) After a drying on a column of polyacrylamide! P10 (Biorad ref. 15011050) the r, c, y ", enL is subjected to a new amplification by PCR for 10 cycles as described above 4J Defermination of the segregation of the plant 3 .., - 1, 1 In a first time the PCR product is removed from the oligonucleotides on a sephac.yl column S400 (rl ~ a "" acia 17-0609-01) then desalted on a column of P10. The sequencing reaction is carried out using the Applied Biosystems kit (ref.
401 628) (373 DNA sequencer) with the primer ar, lisens.
The sequence obtained is identical to the sequence of the SR-p70 cDNA (Example 11.4) with an illse ~, iGn of 198 bp between posilions 217 and 218 (Figure 14). The sequence N-terminal protein deduced ~ e (sequence deno .. ", .ee SR-p70d) is shorter than 49 amino acids with a divergence of the first 13 amino acids (sequence ID
N ~ 13). There is thus co-existence of at least two t, ~ r, s- they are different SR-p70 as already described for the li ~ 3née At r-20 of mice WO 97/28186 PCT ~ R97 / 00214 -B) Cloning of human SR-p70 and demonstration of an AF; Nm coding for a deduced human SR-p70 protein having the same N-terminus as the SR-p70d and a divergence in the C-terminal part 1) Specific amplification of the SR-p7 cDNA by the so-called PCR technique The amplification was carried out as described in EXAMPLE VIIIa from purified ARN of the IMR-32 cells with the pair of amor ~ es of the following composition:
primer meaning: GCG GCC ACG ACC GTG AC (position 160 to 176 sequence ID N ~ 11) anlisens primer: GGC AGC rrG GGT CTC TGG (position 1gg3 to 1976 Figure 6).

After elimination of the excess of primers on column S400 and des ~ lage on column P10 1, u1 of the sample is again subjected to a PCR with the pair arr ~ orces of following composition:
a "~ e sense: TAT CTC GAG CTG TAC GTC GGT GAC CCC
Xho I (posilion 263 to 280 sequence ID
N ~ 11) All ~ Gr ~ e ~ anlisens: ATA ~ CT AGA TCA GTG GAT CTC GGC CTC
Xba I (posi ~ ion 1943 to 1926 Figure 6).

2) Cloning of the product amplifies in plas, pCDNA3 The PCR product obtained in 1) is desalted on a P10 column digested with the enzymes of resl, i.lion Xho I and Xba 1 and then cloned into the plasm "~ i ~ e pCDNA3 as described in EXAMPLE 11.5. Two clones reco, nL, i,) allls are sequenced using the Applied kit Biosystems with specific oligonucleotides of the SR-p70 cDNA.
The first sequence obtained corresponds to the complete sequence of the mRNA
coding for the SR-p70 described in EXAMPLE Vlll.a.ba deduced protein co-uolle 587 amino acids (sequence ID N ~ 13 and Figure 16).
The second sequence obtained is identical to the sequence of the SR-p70d cDNA.
described above but with two deletions of 148 bp and g4 bp between the positions 1049 and 1050 on the one hand and between positions 1188 and 1189 on the other hand (sequence ID
N ~ 14 and Figure 15). The protein sequence deduced from this second sequence reveals a protein having a shorter N-terminal part of 49 amino acids with a divergence in the 13 pre ~ ea amino acids and a divergence of WO 97/28186 PCT ~ R97 / 00214 protein sequence between amino acids 350 and 397 (sequence ID N ~ 15 and FIG.
16) (sequenced: e referred to as SR-p70e). The deduced protein has 506 acids - amines.

C) Demonstration of an mRNA coding for a human SR-p70 protein deduced with a shorter N-terminal e ~ l ~ e 1 ~ Culture of ~; SK-N-SH cells (human neuroblastoma) The cells are cultured as described in the hybridomas 7th edition 1992 ~ ATCC ~ American Type Culture Collection).

2) Preparation of the cDNA and artiplication of the SR-p70 cDNA by the technique known as PCR

These steps are carried out as described in EXEMP ~ E Vill.A with the couple of the following Iposilion:
meaning primer: AGG GGA AGC AGC GAA ACC (position 128 to 145 Figure 17) anti-sense primer: GGC AGC l ~ G GGT CTC TGG (position 1993 to 1976 Figure 6).

Sequencing is performed with the Applied Biosystem kit with primers specific to the cDNA of SR-p70 and reveals two AONc:
a first c ~, ~ s cDNA, ondanl to mRNA coding for SR-p70a a second cDNA having a ~ 8 bp deletion between positions 24 and 25 (sequence ID N ~ 16 and Figure 15).
This deletion cG "~ pr ~ nd the ATG of i., Iliumion of translation of SR-p70a.
ded-read ~ (deno ~ "", ee SR-p70f) of this second cDNA has an ATG initiator of Downstream translation corresponding to an internal ATG of the SR-p70a. The deduced protein COrnpG, therefore: 588 amino acids (sequence ID N ~ 17 and Figure 16) and is truncated 48 amino acids N-te "" of SR-p70a.

Evidence for mRNA encoding human SR-p70b.

1) Cell culture K562 WO 97/28186 PCTn ~ R97 / 00214 -The cells are attenuated as described in the "cataloque of cell lines and hybridomas 7 th edition 1992 "of the ATCC (American Type Cylture Clo" ~ n) 2) Preparation of the cDNA amplification of the cDNA of SR-p70 by the technique of PCR and sequencing.

These steps are carried out as described in EXAMPLE VIII C.

Sequencing reveals two cDNAs:
A first cDNA corresponds to the mRNA encoding SR-p70a and a second CDNA exhibiting a deletion of 94 bp between posilions 1516 and 1517 (sequenceID N ~ 18 and FIG. 15). The deduced protein (called SR-p70b) compo, the 199 amino acids, and has a truncated C-terminal sequence of 137 amino acids relative to SR-p70a with the four divergent amino acids (sequence IDN ~ 1getFigure21).
This cDNA is so similar to that described in EXEMPI EI for monkey SR-p70b.

~ n ~ cl ~ es described in this example (E) CAMPLE Vlll. ABC and D) reveal variants of SR-p70 that are consistent with direct mRNA splicing.
pri ", - L, dns ~ by the SR-P70 gene.
SR-p70a is encoded by mRNA consisting of 14 exons (see EX ~: MPLE VII). It is the pruloid of a woman. The SR-p70b is consecutive to an insertion between the exons 3 and 4 and the absequence of exons 11 and 13. SR-p70f is a consequence of the absence of exon 2. This exe describes the e ~ .is ~ ence of variants of SR-p70 of nonexhaustive manner with a high probability of other variants. Likewise to exist, that of these variants described in this example as well as the SR-p70a is not limited strains in which they have been highlighted.
performed by RT-PCR have shown that these variants are found in the various lined lines In addition, the orientation of the SR-p70f corresponds to a mellium ion.
SR-p70a suggests the possibility of ill (.ialio,) downstream on the mRNA encoding the SR-P70a.
there CA 02245242 l998-07-28 WO 97/28186 PCT ~ R97 / 00214 EXAMPLE IX
. ~
Obtaining a 5 'sequence of human SR-P70a mRNA.
, t 1) Ampl ~ ficat ion of the extremlt ~ ~ 'of the cDNA SR-P70 by PCR
The cell culture and the preparation of total RNA and cDNA are re-read as described in EXAMPLE 1 / 111.1 and 2. The RNA template is hydrolysed by incubation.
minutes at 65 ~ C after adion ~ l, it is 4 IJI EDTA 500 mM and 4 ul NaOH 2N.
The sample is then desalted on P10. The cDNA is aliongé 3 'with a "tail" of dG as described in EXAMPLE 1.3.d in a final volume of 40 1 ~ 1.
After addition of 4, 500 mM EDTA and 4 ~ I NaOH! 2N cDNA is incubated at 65 ~ C for 3 minutes and then desalted on a P10 column. The PCR procedure was performed as described in EXAMPLE V111.3 with 8 μl of cDNA and for 30 cycles.
with the couple of a ", orces of CG" ~ next pOSiliOn primers: CCCCCCCCCCCCCCN (whereNestégalaGAouT) primer anlise: ns: CCATCAGCTCCAGGCTCTC (posilion 1167 to 1149 Figure 6).
After elution of the excess of amines on column S400 and desalting on column P10 1 μl of the hatch is again submitted to a PCR with the pair of following composition:
primers: CCCCCCCCCCCCCCN
antisense primer: CCAGGACAGGCGCAGATG (position 928-911 Figure 6).
The sample again passed on a S400 ~ and a P10 cotonne is submitted to a courtesy, only for 10 cycles during 20 cycles with the following couple:
a ~ Gr ~ esens: CCCCCCCCCCCCCCCN
antisense: CTTGGCGATCTGGCAGTAG (position 503-485 Figure 6).
2) "" i,) at, on sequence 5 'cDNA SR-P70 The sequence is carried out as described in EXAMPLE V111.4). This sequence is a non-coding 5 'of at least 237 bases upstream of the A ~ G of initiation of SR-p70a (Figure 17). By means of this sequence (obtained from the IMR-32 line) with that obtained from line HT-29 in particular (FIG. 6).
two punctuated differences (Figure 17: see bold characters) are {evidence (G - ~ ~ and C ~ T) respectfully positioned at -20 and -30 ATG

CA 02245242 l998-07-28 initiation of SR-p70a (Figures 6 and 17). This variability is located at the level of Exon 2 (Figure 13). It is not excluded that this variability is also found at within a coding phase consecutively to alternative splicing as described in EXAMPLES lll che ~ the mouse and Vlll in humans or else following an initiation of translation on a CTG (as was the case demonstrated for FGFb (Proc Natl Acad Sci USA 1989 86 1836 - 1840).
Similarly, it is not excluded that this variability may have an effect on the translation of the SR-p70 or on the splicing of the primary RNA.
In any case, this variability, probably of Lithuanian origin, can serve as a the marker is at the level g, o ", that (see EXAMPLE Xl) is at the mRNA level (see EXAMPLE X) EXAMPLE X
1) PCR analysis, of the transciplional expression of SR-P70a in the cetlular samples (RT-PCRJ
Cell cultures (SK-N-AS SK-N-MC ItT-29 U-373MG SW480 IMR-32 CHP212) are described as described in example VI.1.a (referenced in caldoc ~ ue ~ 'catalog of cell lines and hy ~ ndo ", as 7th edition" 19 ~ 2 of the ATCC).
The preparation of the cDNA and the PCR test are performed as described in US Pat.
Example Vl11.2 and 3 The pair of primer used is c ~ ", ~ osili ~ n following:
meaning primer: AGGG ~ iACGCAGCGAAACC (posilion 128 to 14 ~ Figure 17) antisense: GGCAGCrrGG ~; ICI ~ G (posiLion 1Q93 to 1976 Figure 6).
The ecl ~ islets are analyzed by elecl ~ pho ~ on a 1% agarose gel and Ethidium Bromide revelation (Figure 18).

The size of the band obtained in the e ~ l, anlil ons corresponds to the expected size (about 2 kb Figures 6 and 17). The intensity of the bands obtained is reproducible.
A re ~ "~ ~ liticalion of 1 ~ I of ecl ~ anillon in the same corldilions during 20 30 cycles make appaidil, ~ a band in each e ~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 2) Detection of the secretion of amplified products After passing samples on columns S400 and P10, the sequencing is carried out 373 sequencer from Applied Biosystems with reference kit 401 628. The 35 primers ~ n ~ séeS are among others the following:
-CA 02245242 l998-07-28 PCT ~ R97 / 00214 figure position AGGGGACGCAGCGAAACC 128to 145 22 CTTGGCGA1-CTGGCAGTAG 503 to 485 6 GATGAGGTGGCTGGCTGGA 677 to659 6 CCATCAGCTCCAGGCTCTC 1167 to 1149 6 TGGTCAGG1 ~ CTGCAGGTG 1605 to 1587 6 GGCAGCTTCiGGTCTCTGG 1993 to 1976 6 No protein differences of SR-p70a were detected. However, the sequences obtained fon1: appa, dîl ~ e a double vafiabiliLe positions -20 and -30 upstream of The ATG of INiLIA ~ iGn SR-p70a (Figures ~ i and 17). This variability, probably of allelic origin, allows to define two classes of l, dnsc.il ~: a first class having a G at position -30 and a C at position -20 (class G-30 / C-20) and a second classie presentan ~ a difference to the two posilions: A in -30 and T in -20 (class A-30, T-20) First class: SK-N-AS, SK-N-MC, Itt-29, U-373MG, SW480.
Second class: IMR-32, CHP212.

EXAMPLE Xl Assay method for the determination of the allelic distribution of the SR-p70 gene in a sample of 10 pe, ~ on This, t: pa, lilic, n "~ 9 is based on the vd, allelic iabilile highlighted in 2 ~ the exe, r, r '25IX and X:
~ Allele G-30l'C-20 having respectively a G and a C at positions -3Q and -20 upstream of the ATG .IIniLi3tion of the SR-p70a ~ All ~ the A-30 / T-20 respectively presenting an A and a T at the same posilions This value can be demonstrated by the use of enzymes of this type.
3 ~ dirre, er) cianl the two alleles (Figure 13) As an example:
~ Enzyme Bpl I prese "ing a cleavage site only on the G-30 / C-20 allele in the area of interest (CQ site encompasses the two variable positions).
Enzyme Styl exhibits a cleavage site only on the A-30 / T-20 allele in the area of the island, el 3 ~

~ PCT FR97 / 00214 .
I) Genomic amplification of exon 2 by PCR
The polymerization reaction is carried out with 500 ng of purified genomic DNA
in 50 ~ final ul with the conditions described in Example Vl11.3.
The pair of primers has the following composition:

meaning primer: CACCTACTCCAGGGATGC (position 1 to 18 Figure 13) antisense primer: AGGAAAATAGAAGCGTCAGTC (position 833 to 813 Figure 13).

The reaction is carried out for 30 cycles as described in EXAMPLE VI11.3.
After removal of excess primer on an S400 column and drying on a colollne P10 1 ~ I of the sc, l "~ n is a", plirié again during 25 cycles in the same condilions with the following pair of primers:

meaning primer: CAGGCCCACrrGCCTGCC (position 25 to 32 Figure.13) antisense primer: CTG ~ CCCCAAGCTGATGAG (position 506-488 Figure 13).

The insect products are subjected to an agarose gel on a 1% agarose gel.
(Figure 19-A).
2) Digestion with Enzyme ~ Styl The samples are preatabte ~ ~ ~ es on a P10 column and then digested with the enzyme re ~ lioli) Styl (BRL 15442-015) in the following "~ ~" - ~
Tris HCl pH 8 50 mM NaCl 100 mM MgCl 2 10 mM at 37 ~ C for 30 min. The ~ lige products, ~ ion are analyzed by electrophoresis on a ge ~ agarose 1%
2 ~ (~ a. "Pon TAE) The rev ~ i ~ tion is achieved by cclăralion bromide ethidium (Figure ~ 9-B ~.

A band of 482 base pairs cara ~ lense the allele G-30 / C-20 ~ Figures 13 and 19).
The prese ~ ~ c ~ a band of 376 base pairs and a band of 106 pairs of bases c ~, a ~ l ~ isenl the A-301T-20 allele (allele presenla, -L a Styl cleavage site).
On the sample, the sample size of 10 pe, aons, 2 pe ~ ss, have alleles G-30 / C-20 and A-30 / T-20 the remaining 8 individuals being homozygous with the G-30 / C-20 allele.
The study of a new sample of 9 persons showed 3 persons heterozy ~ otes presenlan ~ the alleles G-30 / C-20 and A-30 / T-20 the 6 other people andanthomozygotes for G-30 / C-20 allele.

WO 97/28186 PCT ~ R97 / 00214 .

EXAMPLE Xll Revision of transfol "assay, alion of line SK-N-AS by lld. ~ Sfe ~ lion with I ~ SR-p70 cDNA.

The expression vector used is described in EXAMPLE 11.5 and schematized in Figure 15. The method used is that called calcium phosphate described by Graham et al. (Virology 1973, 4, 2, 536-539). The line is sown at the rate of 5.105 cells per 6 cm dish of dia- "eel in 5 ml of the medium described in Example 1. The cells are cultured at 37.degree. C. and at .about.
night. The medium of transr ~~ i ~ n is prepared from ", anièrt: following: the following mixture is made ~ by adding in the order 1 ml of HERS buffer (NaCl 8 mg / ml, KCl 370, ug / ml, Na2HPO4-2H20 t25, ug / ml, De ~ l, 1 mg / ml, Hepes pH 7 , 05 5 mg / ml), 10 ~ ug from plasmid to transfect and 50 μl of 2.5 M CaCl 2 added dropwise. The middle of transfection is left for 30 minutes at a temperature, then it is added dropwise to the storytelling environment in the culture box. The cells are incubated at 6 hours at 37 ~ C / 5% CO2. After stirring, from the middle, 5 ml of fresh medium containing ~% of fetal bovine serum are added after 48 hours at 37 ~ C / 5% CO2, the cells are rinsed with P ~ 3S, peeled by try, ~, ~ i "isaliol-, diluted in 10 ml of medium culture (5% fetal bovine serum) and spread in a 10 cm dish of lid, (Dilution can be adjusted to fon ~, lion of ~ the erricacile of t, d "sreclion).
new incuhation for 10 hours (the time that ~ e "L ~ the adhere), the cells have passed in the 5th century, GII paradjunction of 3 G418 to the final decision of 600 l ~ g / ml genélic equivalent for 15 to 21 days (the medium is changed every 30s).
The clones obtained are then rinsed with PBSI fixed with 70% ethanol, dried, colored.
with 1% crystal violet, then comp ~ - ~ hil;
Four of these were made in duplicate:
- pld ~ "~; pCDNA3 without insert Plasmid pCDNA3 / SR-p70 Contains Human SR-P70a cDNA
pCDNA3 / SR-p70 mut plasmid, the cDNA-SR-p70a exhibiting mutation at posion 293 AA (R ~ H) which is analogous to mutation 273 (R -H ~ in the DNA binding domain of p53 - control without plasmid.
3 ~
The result is expressed in number of clones per box.

WO 97/28186 PCT ~ FR97 / 00214 Experience 1Experience 2Middle pCDNA3 172,353,262 pCDNA3 / SR-p70 13 8 10 pCDNA3 / SR-p70 mut 92 87 89 Absence of pta ~ "l: de 1 3 2 The number of clones obtained by transfection with the plasmid pCDNA3 / SR-p70 is 25 fold lower than the number of clones obtained with the pCDNA3 control and 9 times less than the number of clones obtained with pcDNA3 / SR-p70 Mut indicating a mortality or cell division arrest ~ cells transfected with SR-p70 cDNA. This result is not the consequence of a toxicity in view of the clones obtained with the SR-p70 cDNA mute but probably an apoplosis as has been demonstrated for p53 protein (Koshland et al., Science 1 9g3 262 1953 ~ 1981).

EXEMP ~ E Xlil Biological Role of the pfuLei .. e SR-p70.
Structure homology between the DNA binding domain of p53 and the region The central point of the SR-p70 allows the interception of the SR-p70 to be a dellnnsc factor (see Figures 1 and 2). Indeed the p53 (393 amino acids) is constituted The N-terminal region (1-91 amino acids) is flusie ~ rs It is linked in the activation of the ls ansc, ipLion and conlienl sites interaction to dirter ~ r) cellular and viral proteins. The central part (amino acids 92 to 292) allows fixation to specific DNA sequences located in regions Some of these genes, such as p53 mutations, inactivate p53.
are located in this region) it also has many sites interaction with viral proteins that inhibit its activity. Finally the 1 û0 The last amino acids of p53 are responsible for its oligomerization as well as of the regulation of cell ~ ci ~ Hainaut P. Current Opinion in Oncology 1995 7 76-82;
ProkocimerM Blood 1994 84n ~ 8 2391-2411).
The sequence of the sequence between p53 and SR-p70 is ## EQU1 ## where "Amino acids i" directly plotted in the DNA interaction suggesting that SR-p70 binds to p53 sites on DNA. These amino acids =

CA 02245242 l998-07-28 WO 97/28186 PCT ~ R97 / 00214 .. 3g correspond exactly to what we call hot spots, amino acids frequently mutated in human tumors (SWiSS PROT: SW: P53_human and Prokocimer M., Blood, 1994, 84, No. 8, 2391-2411). From this homology, we can deduce that the SR-p70 protein controls the activity of genes regulated by p53, Either independently of it or by forming heterooligomers with this last.
Consequently, like p53, the SR-p70 gene products must be involved in the control and regulation of the cell cycle causing arrest of the cycle (", G", initiated or definitive), and the implementation of programs such as:
DNA repair, administration or cell death. The existence of activities 'p53-like' was strongly presentiated with the detection in mice p53 ~ / ~, DNA repair activities and cell death in response to ionizing radiation (Strasser et al., Cell, 1994, 79, 329-339). The authors of the The present invention has localized the human SR-p70 gene in the telomeric reion ion of short arm of chromosome 1, precisely in 1 p36.2-36.3, the smallest region deletion (SRO) common to a ~ ~ aio, it ~ neu, oblasLomes and other types of tumors (melanomas and fungi) (White et al., PNAS, 1995, 92, 5520-5-24).
This region of loss of heterozygosity (LOH) delimits the locus of a suppressor gene The loss of activity would be the cause of tumor formation. It is It is important to note that this re ~ ion is also subject to maternal "alien") is usually lost in neuroblastoma presenting the deletion 1p36 (without alplicion of N-Myc) (Caron et al., Hum Mol.
Gen., 19g5, 4, 535-539) The SR-p70 wild-type gene introduced and expressed in These are neues, which allow me to reversion of their transition. The loss of this activity ~ anti-Gnco9én ~ ~ e is aCsGr é ~ the development of the tumor. The region 1 p36 presented a syngenic hGr with the distal segment of the cl ~ ro, noso.) ~ e 4 mouse. In this re ~ ion was lo ~ - 'se the curfy tail gene (cf) (Beier et al., Mal, Ill, alian Genome, 1995, 6, 269-272), published in the English language.
congenital neural tube (MTN: spida bifda, anencephaly ...). The mouse is the best animal study model of these, -, ~ lto, ll ~ alions. It is accepted that these , ualrulllld ~ iGnsl result from ano, ll ~ 'es of cell proliferation Given the the nature of the SR-p70 gene and its chromosomal location, one of the hypotheses is that the SR-p70 could be the human, and that, as such, the detection of early mutations and allergic ano, aliases related to this gene should be allow, for example, the applicability of the identification of people at risk (0.5-1% of newborns with MTN), and the implementation of Preventffs (Neumann et al., Nature Genetics 1994 6 357-362, Di Vinci et al J. Int.
Cancer 1994 59 422-426; Moll et al., PNAS 1995 92 ~ 4407-4411; Chen and a /., Development 1995 121, 681-691).

EXAMPLE XIV

Allelic study of the SR-p70 gene.
The GC and AT alleles are easily identified by the Styl esclusion of the PCR products of exon 2 (see example Xl). It was thus possible to determine in individuals GC / AT heterozygotes and neuroblastoma tumor carriers the SR-p70 allele lost (GC or AT) despite the presence of healthy contaminating tissue.
In a su "way, renanla when the same analysis is performed on ur RNA alone allele is demonstrated independently of the presence or absence of a deletion and even more surprising despite the presence of healthy tissue. This suggests that the imprint (expression dirrer ~: "lielles of the two alleles) would also exisler in the conldl "inant fabric.
To verify this, the same analysis was repeated on RNA from the cells of healthy individuals heterozygous GC / A ~. Only one of two types of Lrdns ~
has been detected e ~ 'en ent in these cel ~ Qs This result confirms the observation made on Tumor samples for the existence of a genetically modified genetic system for the SR-p70 gene.
The conclusions of this discovery are i, ll ~ Gildl, las since it makes it possible to apply A single sporadic mutation inactivating the SR-p70 active allele will result in a loss.
of activity and this poole, ~ éle "- ~ nt in all tissues.
The absence of precise doses on the SR-p70 metabolic function does not allow to quantify the extent of this loss of SR-p70 activity for the cell.
N ~ anrl, G;., S its strong homology with the pr ~, t8; tumor suppressor p53 as well That the den, onal, determines that SR-p70 is a transcription factor C: ~ p ~ Q to use the, c, o ", oLaur P21 ~ suggests a role of this protein in the control of cyrlecellu 'lire and in the dirré, ~ ncialio"
Knudson and Meadows 1980 (New Eng J. Med 302: 1254-56) consider the neu, ohl ~ sl ~ rs IV-S as a collection of unmelted cells of the ridge Neural mutation carrying a mutation, which is rare with their clonality, is normal.

CA 02245242 l998-07-28 WO 97/28186 PCTA ~ R97 / 00214 It is conceivable that the loss of SR-p70 activity just like the loss of control p53 on the cell cycle promotes the appearance of cellular abnormalities such as aneuploidy amplification (described in the case of neu ~ o ~ lu ", es) and other genetic rearrangements that can cause transron "cell aLion (Livingstone et al. 19 ~ 2 Cell 71: 923-25; Yin et al. 1992 Cell 72: 937-48; Cross et al. 1995 Science 267: 1353-! ~ I6; Fukasawa et al. 1996 Science 271: 1744-47). The neuroblastomas could therefore originate from a loss of activity temporary or definitive of the SR-p70, thus favoring the presentation of events oncogenic and therefore proy, ~: tumor ssion.
In the case of constitutional deposition 1 p36 described by Biegel et al 1993 (Am. J.
Hmm. Broom. 52: 176-82), there is good app ~ .ilio "neuroblastoma IV-S and the gene concerned is NBS-1 (SR-p70).
In conclusion what is described for neuroblastomas could also be apply to other types of tumors, particularly those that have been associated with , ~ n, anie, enls of the e ~ cl, izr "island of the short arm of ~ ulllosollle 1 (report 2 illte" ~ a ~ iGnal workshop on human chr 1 mapping 1995 Cytogenetics and Cell Genet. 72: 113-154).
On a therapeutic level, the use of SR-p70 in tumor administration should lead to avoiding the use of multiple agents in the form of risks of cellular impairment by these products and to prefer them to non-mutagenic substances that stimulate differentiation.
On the other hand, the frequency of app ~ ion of GC and AT alleles is as follows: in the pop ~ tion Frequency (AT) = 0. 15 and on a sample of 25 patients (neurohl-sto ", es) F (AT) = 0.30, which indicate that the AT allele might be a factor of p, e ~; spo ~; ~; on.

WO 97t28186 42 PCTAFR97 / 00214 LIST OF SEQ ~ INCES

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(A) NAME / KEY: CDS
(B) LOCATION: 156..2066 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:
TGCCTCCCCG CCCGCGCACC CGCCCC ~ - ~ GG C ~ l ~. ~, CC TGCGA ~ GGGG ACGCAGCGAA 60 GCCGGGGCCC GCGCCAGGCC GGCCGG ~ ACG GACGCCGATG CCCGGAGCTG CGACGGCTGC 120 AGAGCGAGCT GCC ~ lCG ~ AG GCcG ~. ~. ~ GGAAG ATG GCC CAG TCC ACC ACC 173 Met Ala Thr Thr Thr Thr Ser Pro A ~ p Gly Gly Thr Thr Phe Glu Hi ~ Leu Trp Ser Ser Leu Glu Pro A ~ p Ser Thr Tyr Phe A ~ p Leu Pro Gln Ser Ser Arg Gly Asn Asn Glu Val Val Gly Gly Thr A- ~ p Ser Ser Met A3p Val Phe His Leu Glu Gly Met Thr Thr Ser Ser Met Ala Gln Phe Asn Ser Leu Leu ser Ser Thr Met A ~ p Gln Ser Ser Ser Ala Ser Ala Ser Pro Ser Ser Pro CA 0224 ~ 242 1998-07-28 WO 97/28186 ~ 3 PCT ~ R97 / 00214 CCG GAG CAC GCC GCC AGC GTG CcC ACC cAT TCA cCC TAC GCA CAG CcC 461 Pro Glu Hi ~ Ala Ala Ser Pro Val Pro His Ser Pro Pro Ala Gln Pro AGC CBT ACC TTC GAC ACC ATG TCG CCC GCG CCT GTC ATC ccC TCc AAC S 09 Ser Ser Thr Phe P ~ ~ p Thr Ser Ser Pro Ala Pro Val Pro Island Ser Asn ACC GAC TAT CCC G: GA CCC CAC CAC TTC GAG GTC ACT TTC CAG CAG TCC S57 Asp Asp Tyr Pro Gly Pro EIi ~ His Phe Glu Val Thr Phe Gln Gln Ser Ser Thr Ala Ser Lys Ser Ala Thr Thrp Thr Tyr Ser Pro Leu Leu Ly ~ Lys 135 140 145 lS0 Leu Tyr Cys Gln Island Ala Lys Thr cy9 Pro Island Gln Ile Lys Val Ser Pro Ala Pro Pro Gly Thr Ala Arg Island Ala Pro Met Val Tyr Lys AAG GcG GAG CAC GTG ACC GAC ATC GTG AGC CGC TGC CCC AAC CAC GAG 749 LyY Ala Glu Hi ~ Asp Val Asp Asp Val Lys Arg Cy ~ Pro Asn His Glu Leu Gly Arg Asp Phe Asn Glu Gly Gln Ser Ala Pro Ala Ser His Leu Arg Val Glu Island Gly Asn Asn Leu Ser Gln Tyr Valley A ~ p Asp Pro Val Thr Gly Arg Gln Ser Val Val Val Pro Glu Pro Gln Pro Gln Thr Glu Phe Thr Thr Ile Leu Tire Aqn Phe Met Cy ~ Asn Ser Ser Cys GTG GGG GGC ATG AAC CGA CG & CCC ATC CTC ATC ATC ATC ACC CTG GAG 989 Val Gly Gly Met Arg Arg Pro A3n Island Leu Ile Ile Ile Thr Leu Glu Thr Arg Aqp Gly Gln Val Leu Gly Arg Arg Ser Phe Glu Gly Arg Ile 280,285,290 CyY Ala Cys Pro Gly Arg A ~ p Arg Ly ~ Ala A-lp Glu A ~ p Hi C Tyr Arg Glu Gln Gln Ala Leu A ~ n Glu Ser Ser Ala Ly ~ A ~ n Gly Ala Ala Ser Lys Arg Ala Phe Lys Pro Gln Ser Pro Ala Pro Pro Ala Leu Gly Pro Gly Val Ly! L Lys Arg Arg Hi ~ Gly Asp Glu A ~ p Thr Tyr Tyr Leu Gln GTG CGA GGC CGC GA ~ AAC TTC G ~ G ATC CTG ATG AAG CTG AAG GAG AGC 1277 Val Arg Gly Arg Glu Asn Phe Glu Ile Leu Met Ly ~ Leu Ly ~ Glu Ser CA 0224 ~ 242 1998-07-28 WO 97/28186 1 ~ PCT ~ F ~ 97/00214 The Glu Leu Leu Met Glu Leu Val Pro Pro Gln Leu Val Asp Asp Ser Tyr ALg 375 380 38 ~ 390 Gln Gln Gln Gln Gln Leu Gln Arg Pro Ser Ser Leu Gln Pro Pro Ser Tyr Gly Pro Val Leu Pro Ser A ~ n Ly ~ Hi Val ~ Gly Gly Val Asn 410 ~ 15,420 A ~ G CTG CCC TCC GTC AAC CAG CTG GTG GGC CAG CCT CCC CCG CAC AGC 1469 Ly ~ Leu Pro Ser Val Asn Gln Leu Val Pro Gly Gln Pro Pro His Ser Ser Ala Ala Thr Pro Asn Leu Gly Pro Val Gly Ser Gly Met Leu Asn AAC CAC GGC CAC GCA GTG CCA GCC AAC AGC GAG ATG ACC AGC AGC CAC lS65 A ~ n Hi. ~ Gly Hia Ala Val Pro Ala A ~ n Ser Glu Met Thr Ser Ser Hi ~

Gly Thr Gln Ser Met Ser Val Ser Gly Hi ~ Cy ~ Thr Pro Pro Pro Pro Tyr Hls Ala A ~ p Ser Ser Ser Leu Val Ser Phe Leu Gly Leu Leu Gly Cy ~

Pro A ~ n CyY Island Glu Tire Phe Thr Ser Gln Gly Leu Gln Ser Tire Island Hi ~ Leu Gln A ~ n Leu Thr Ile Glu A p Leu Leu Gly Ala Ly ~ Ile Pro 520,525,530 Glu Gln Tyr Arg Met Thr Ile Trp Arg Gly Leu Asp Gln Leu Ly ~ Gln 535,540,545,550 Gly Hi. ~ A ~ p Tyr Gly Ala Ala Ala Gln Gln Leu Leu Arg Ser Ser A ~ n 555,560,565 Ala Ala Ala Ser Island Gly Gly Ser Gly Glu Gln Arg Gln Arg Val Met Glu Ala Val Hi ~ Phe Arg Arg Arg Hi3 Thr Island Thr Island Pro A ~ n Arg Gly Gly Pro Gly Ala Gly Pro A ~ p Glu Trp Ala Asp Phe Gly 600,605,610 Phe A ~ p Leu Pro A ~ p Cy- ~ Ly ~ Ala Arg Lys Gln Pro Ile Ly ~ Glu Glu Phe Thr Glu Ala Glu Hi Island ~

CACCGCCCAG AGACCCAGGC CGCCTCGCTC LC ~ l ~ l ~ 'l GTCCAAAACT ~ C ~ CCGGAG 2156 GCAGGGCCTC CAGGC. ~ LGC CCGGGGAAAG GCAAGGTCCG GCCCATGCCC CGGCACCTCA 2216 -CA 02245242 l998-07-28 WO 97/28186 45 PCT ~ R97 / 00214 CCGGCCCC ~ G GAGAGGCCCA GCCACCAAAG CCGCCTGCGG ACAGCCTGAG TCACCTGCAG 2276 CCTCCAGGCC TCATCCTAGA GACTC ~ GTCA TCTGCCGATC AAGCAAGGTC CTTCCAGAGG 2456 AGAGTGGTGA GCAGCCA ~ GC GACTGTGTCT GAAACACCGT GCATTTTCAG GGAATGTccc 2576 ATTCTGCGGG ACCGCCTCCT TCCTGCCCCT AACAACCACC AAA ~ l ~ lLGC TGAAATTGGA 2696 AGTGCCCCTC AGCCTGGCCA CAGTCACCTC TC ~ LLG ~ A ACCCTGGGCA GAAAGGGACA 2816 GCCTGTCCTT AGAGGACCGG AAATTGTCAA TATTTGATAA AATGATACCC ~ IlL ~ lAC 2874 (2) INFORMATION FOR SEQ ID NO: 2:
(i) CHARACTERISTICS OF THE SEQUENCE:
~ A) LENGTH: 637 amino acids (B) TYPE: amino acid (D) COr ~ FIGURATION: linear (ii) TYPE OF MOLECULE: protein (xi) D ~ SCRIPTION OF THE SEQUENC ~: SEQ ID NO: 2:
Ala Gln Ser Hr Thr Thr Ser Pro Asp Gly Gly Thr Thr Phe Glu ~ S 10 15 ~ is Leu Trp Ser Ser Leu Glu Pro A ~ p Ser Thr Tyr Phe A ~ p Leu Pro Gln Ser Ser Arg Gly Asn A ~ n Glu Val Gly Gly Thr A ~ p Ser Ser Met Asp Val Phe His Leu Glu Gly Met Thr Thr Ser Val Met Ala Gln Phe Asn Leu Ser Ser Ser Ser Met Asp Gln Ser Ser Ser Arg Ala Ala ~ er Pro Ala Ser Pro Thr Pro Glu His Ala Pro Ala Ser Pro Thr His ~ Pro Pro Ala Gln Pro Ser Pro Ser Thr Phe A ~ Thr P Met Ser Pro Ala Pro Pro Island Pro Ser A ~ n Thr A ~ p Pro Tyr Pro Hi Gly ~ His Phe Glu Val Thr Phe Gln Ser Gln Ser Ser Ala Ly ~ Ser Ser Thr Thrax Ser Pro Leu Leu Ly Ly Leu Tyr Cy ~ Gln Ile Ala Lys Thr Cys Pro ~ Gln Ile Lys Val Ser Pro Ala Pro Pro Gly Thr Ala Arg Island ~ Met Pro Val Ly Ly ~ Ly ~ Ala Hi Glu ~ Val Thr Asp Ile Val Lys 180 to 185 ~ 190 Arg Cy ~ Pro Asn His Glu Leu Gly Arg Asp Phe A- ~ n Glu Gly Gln Ser CA 0224 ~ 242 l998-07-28 WO 97/28186 46 PCTAFR97 / 0 ~ 214 Ala Pro Ala Ser His Leu Arg Island Glu Gly Gly Asn Asn Leu Ser Gln Tyr Val Asp Asp Pro Val Thr Gly Arg Gln Val Val Val Val Pro Tyr Glu Pro Pro Gln Val Gly Thr Glu ehe Thr Thr Leu Tyr Asn Phe 2g5 250 255 Met Cys Asn Ser Ser Cys Val Gly Gly Met Asn Arg Arg Pro Ile Leu 260,265,270 Ile Ile Thr Island Leu Glu Thr Asp Asp Gly Gln Val Leu Gly Arg Arg Ser Phe Glu Gly Arg Cys Ala Cy ~ Pro Gly Arg Arg Arg Ala Ala 290,295,300 A3p Glu Asp His Tyr Arg Glu Gln Gln Ala Leu Asn Glu Ser Ser Ala Lys Asn Gly Ala Ala Ser Lya Arg Ala Phe Lys Gln Pro Ser Pro Ala Val Pro Ala Leu Gly Pro Gly Val Lys Lys Arg Arg His Gly Asp Glu 340 345 3 ~ 0 Asp Thr Tyr Tyr Leu Gln Val Arg Gly Arg Glu A. ~ n Phe Glu Ile Leu Met Lys Leu Lys Glu Ser Glu Leu Leu Glu Leu Gln Pro Gln Pro 370,375,380 Leu Val Asp Ser Tyr Arg Gln Gln Gln Gln Leu Leu Gln Arg Pro Ser Hi3 Leuk Pro Gln Pro Ser Gly Pro Pro Leuk Ser Ser Pro Asn Ly3 Val His Gly Gly Val Asn Lys Leu Pro Val Val Asn Gln Leu Val Gly 420,425,430 Gln Pro Pro Hi-Ser Pro Ala Ala Thr Pro Asn Leu Pro Gly Pro Gly ser Gly Met Leu AYn A ~ n Hiq Gly Hi ~ Ala Val Pro Ala Asn Ser Glu Met Thr Ser Ser Ser Hia Gly Gln Ser Met Ser Val Ser Gly Ser His Pro Pro Cys Thr Pro Pro Hi ~ Ala A! ~ Pro Ser Pro Ser Val Phe Leu Thr Gly Leu Gly Cya Pro Asn Cya Island G1U Tyr Phe Thr Ser Gln Gly Leu Gln Ser Tyr Island Hi ~ Leu Gln Aan Leu Thr Ile Glu Asp Leu Gly Ala Leu Lys Pro Island G u Gln Tyr Arg Met Thr Ile Trp Arg Gly Leu Gln Asp Leu Lys G n Gly Hi ~ Asp Tyr Gly Ala ~ the Ala Gln Gln Leu Leu Arg Ser Ser Asn Ala Ala Ala Ile Ser Ile Gly Gly Ser Gly r Gln Leu Gln Arg Gln Arg Val Glu Ala Val Arg Arg HiY Thr Thr Island Pro Island Asn Arg Gly Pro Gly Pro Gly Ala Gly Pro 595,600,605 Glu Trp Ala Asp ehe Gly Phe Asp Leu Pro Asp Cy ~ Ly ~ Ala Arg Ly3 Gln Pro Lys Island Glu Glu Phe Thr Ala Glu Glu Island His (2) INFORMATION FOR SEQ ID NO: 3:
(i) CHARACTERISTICS OF THE S ~ QU ~ NCE.
(A) LOI ~ GUEUR: 2034 pair ~ of bases (B) TYPE: nucleic acid (C) NO2 ~ RE OF BRINS: double (D) CONFIGURATION: linealrc (ii) TYPE OF MOL ~ CULE: cDNA
(vi) ORIGIN:
(A) ORGANIZATION: Cebus Apella (ix) ADDITIONAL CHARACTERISTICS:
(A) NAME / KEY: CDS
(B) LOCATION: 156..1652 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:
TGCCTCCCCG CCCGCG ~ .CC CGCCCCGAGG C - L ~ 1 ~ 1CC TGCGAAGGGG ACGCAGCGAA 60 GCCGGGGCCC GCGCCA.GGCC GGCCGGGACG GACGCCGATG CCCGGAGCTG CGACGGCTGC 120 AGAGCGAGCT GCCCTCGGAG GCCG ~ l ~ A GGAAG ATG GCC AGC TCC ACC ACC 173 Met Ala Thr Thr Thr Thr Ser Pro A ~ p Gly Gly Thr Thr Phe Glu Hi ~ Leu Trp Ser Ser Leu Glu Pro A ~ p Ser Thr Tyr Phe Asp Leu Pro Gln Ser Ser Arg Gly Asn A ~ n Glu Val Val Gly Gly Thr A ~ p Ser Ser Met A ~ p Val Phe Hi ~ Leu Glu Gly Met Thr Thr Ser Val Met Ala Gln Phe A ~ n The ~ Leu Ser Ser Thr Met A ~ p Gln M ~ t Ser Ser Arg Ala Ser Ala Ser Ser Pro Tyr Thr Pro Glu Hi ~ Ala A] .a Pro Ser Ser Pro His Ser Pro Pro Ala Pro Gln Pro AGC TCC ACC TTC G ~ -C ACC ATG TCG CCC GCG CCT GTC ATC CCC TCC AAC 509 Ser Ser Thr Phe A ~ p Thr Ser Ser Pro Pro Ala Pro Val Pro Ser Asn Thr A ~ p Tyr Pro Gly Pro His Hi ~ Phe Glu Val Thr Phe Gln Gln Ser CA 0224 ~ 242 1998-07-28 WO 97/28186 48 PCT ~ FR97 / 00214 Ser Thr Ala Lys Ser Ala Thr Trp Thr Tyr Ser Pro Leu Leu Lys Lys 135 140 '145 150 Leu Tyr Cy ~ Gln Ile Ala Lys Thr Cy ~ Pro Ile Gln Ile Lys Val ser Pro Ala Pro Pro Gly Thr Ala Arg Island Ala Pro Met Val Tyr Lys Lys Ala Glu His Val Thr Asp Val Lys Island Arg Cy9 Pro Asn Hi ~ Glu Leu Gly Arg Asp Phe Asn Glu Gly Gln Ser Ala Pro Ala Ser His Leu Ile Arg Val Glu Gly Aqn Asn Leu Ser Gln Tire Val Asp A ~ p Pro Val Thr Gly Arg Gln Ser Val Val Val Pro Glu Pro Gln Pro Gln Thr Glu Phe Thr Thr Ile Leu Tyr A ~ n Phe Cy3 Met Asn Ser Ser Cys Val Gly Gly Met Asn Arg Arg Pro Ile Leu Ile Ile Ile Thr Leu Glu Thr Arg Asp Gly Gln Val Leu Gly Arg Arg Ser Phe Glu Gly Arg Ile 280 285 29 ~

Cys Ala Cyq Pro Gly Arg Asp Arg Ly ~ Ala A ~ p Glu A3p His Tyr Arg Glu Gln Gln Ala Leu A Glu Ser Ser Ala Lys Asn Gly Ala Ala Ser Lys Arg Ala Phe Ly Gln Pro Pro Ala Pro Pro Ala Leu Gly Pro Gly Val Lyq Ly ~ Arg Arg Hi ~ Gly A- ~ p Glu A: sp Tyr Tyr Tyr Gln Gln Val Arg Gly Arg Glu A ~ n Phe Glu Ile Leu Met Lys Leu Ly3 Glu Ser Leu Glu Leu Met Glu Leu Val Pro Pro Gln Leu Val A ~ p Ser Tyr Arg Gln Gln Gln Gln Leu Leu Gln Arg Ser Ser Pro ~ Leu Gln Pro Pro Ser Tyr Gly Pro Val Leu Pro Ser Met Asn Lys Val His Gly Gly Val A ~ n WO 97/28186 PCTrFR97 / 00214 Lys Leu Pro Ser Asn Val Gln Leu Val Gly Pro Gln Pro Pro His Ser 425,430. 435 Ala Ala Thr Pro A ~ n Leu Gly Pro Val Gly Ser Gly Met Leu A ~ n AAC CAC GGC CAC GCA GTG CCA GCC AAC AGC GAG ATG ACC AGC AGC cAC 1565 Asn Hi ~ Gly His Ala Val Pro Ala Asn Ser Glu Met Thr Ser Ser His Gly Thr Gln Ser Will Val Ser Gly Ser Hi ~ Pro Cys Pro Pro Pro Tyr Hls Ala Asp Ser Pro Ser Val Arg Thr Trp Gly Pro CGGCGCCGCC GCGCAGCAGC TGCTCCGCTC CAGCAACGCG GCCGCCATTT CCATCG & CGG 1782 CGAGATCCAC TGAGGG ~ GCCG GGCCCAGCCA GAGCCTGTGC CACCGCCCAG AGACCCAGGC 2022 (2) IW ~ ORMATION} 'OUR SEQ ID NO: 4:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 499 acid- ~ amine3 (B) TYPE: amino acid ~
(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: protein ¢
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:
Ala Gln Thr Thr Thr Thr Thr Pro A Gly Gly Thr Thr Phe Glu ~ iY Leu Trp Ser Ser Leu Glu Pro Asp Ser Thr Tyr Phe A ~ p Leu Pro Gln Ser Ser Arg G.ly A ~ n A ~ n Glu Val Gly Gly Thr A ~ p Ser Ser Met A ~ p Val Phe H.ia Leu Glu Gly Met Thr Thr Ser Val Met Ala Gln Phe A ~ n Leu Leu ser Ser Ser Met A ~ p Gln Ser Ser Ser Arg Ala Ala ~ er Pro Ala Ser Pro Thr Pro Glu Hia Ala Ser Pro Pro Thr Hi ~ Ala Ser ~ 5 90 95 ~ Pro Pro Ala Gln Pro Ser Pro Ser Thr Phe A ~ Thr P Met Ser Pro Ala Pro Pro Island Pro Ser AYn Thr Asp Pro Tyr Pro Gly Pro His Hi ~ Phe Glu Val Thr Phe Gln Ser Gln Ser Thr Ala Lya Ser Ala ~ hr Trp Thr Tyr CA 0224 ~ 242 1998-07-28 WO 97/28186 n / a PCTrFR97 / 00214 Ser Pro Leu Leu Lys Lys Leu Tyr Cys Gln Ile Ala Lys Thr Cys ero ~ Gln Ile Lys Val Ser Pro Ala Pro Pro Gly Thr Ala Arg Island ~ Met Pro Val Lya Ly Ly3 Ala Glu His Val Thr Asp Island Val Lys Arg Cys Pro Asn His Glu Leu Gly Arg Asp Phe Asn Glu Gly Gln Ser Ala Pro Ala Ser His Leu Isle Arg Val Glu Gly Asn A ~ n Leu Ser Gln 210 to 215 ~ 220 Tyr Val Asp Asp Pro Val Thr Gly Arg Val Gln Val Val Val Pro Tyr ~ Pro Pro Gln Gln Val Gly Thr Glu Phe Thr Thr Island Leu Tyr Asn Phe ~ e ~ Cys A3n Ser Ser Cys Val Gly Gly Met Asn Arg Arg Pro Ile Leu 260,265,270 Ile Ile Thr Island Leu Glu Thr Asp Asp Gly Gln Val Leu Gly Arg Arg Ser Phe Glu Gly Arg Island Cys Ala Cy Pro Gly Arg A ~ p Arg Lys Ala 290,295,300 Asp Glu Asp His Tyr Arg Glu Gln Gln Ala Leu Asn Glu Ser Ser Ala ~ y ~ Asn Gly Ala Ala Ser Lya Arg Ala Phe Ly ~ Gln Ser Pro Pro Ala ~ al Pro Ala Leu Gly Pro Gly Val Lys Lys Arg Arg His Gly Asp Glu 340,345,350 Aap Thr Tyr Tyr Leu Gln Val Arg Gly Arg Glu Asn Phe Glu Ile Leu Met Ly ~ Leu Ly ~ Glu Ser Leu G1U Leu Met Glu Leu Val Pro Gln Pro 370,375,380 Leu Val A ~ p Ser Tyr Arg Gln Gln Gln Gln Gln Leu Leu Gln Arg Pro Ser ~ i ~ Leu Gln Pro Ser Ser Pro Gly Pro Leu Val Pro Pro Met Aan Lys ~ al Hi ~ Gly Gly Val Asn Ly ~ Leu Pro Ser Aan Val Gln Leu Val Gly 420,425,430 Gln Pro Pro Pro His Ser Ser Ala Pro Ala Thr Asn Leu Gly Pro Val Gly Ser Gly Met Leu Aqn Aan HiY Gly Hi ~ Ala Val Pro Ala A ~ n Ser Glu Met Thr Ser Hi Ser Gly Thr Gln Ser Met Ser Val Ser Gly Ser ~ ys Pro Pro Thr Pro Pro Hi Hi ~ Ala A ~ Pro p Pro Leu Val Arg Thr ~ rp Gly Pro ~ 2) INFORMATION FOR S ~ Q ID NO: S:

- =
CA 02245242 l998-07-28 WO 97/28186 ~ I PCT ~ FR97t00214 ~ (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 2156 base pairs ~
(B) TYPE: Acid Nucleus (C) NUMBER OF BRINS: double (D) CONFIGURATION: linear (il) TYPE OF MOLECULE: cDNA
(vi) ORIGIN:
(A) ORGANIZATION: Homo sapiens (ix) ADDITIONAL CHARACTERISTICS:
(A) NOI ~ / KEY: CDS
(B) LOCATION: 33..1940 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:

Ala Gln Ser Ser Thr Ala Thr TCC CCT GAT GGG GGC ACC ACG TTT GAG cAc CTC TGG AGC TCT CTG GAA 101 Ser Pro Asp Gly Gly Thr Thr Phe Glu His Leu Trp Ser Ser Leu Glu Pro A3p Ser Thr the Yr Phe Asp Pro Leu Gln Ser Ser Arg Gly Asn Asn Glu Val Val Gly Gly Thr Asp Ser ser Met A ~ p Val Phe His Leu Glu Gly Met Thr Thr Ser Val Met Ala Gln Phe A ~ n Leu Leu Ser Ser Thr Met Asp Ser Ser Ser Arg Ala Ala Pro Al Ser Pro Tyr Thr Pro Glu His Ala Ala Ser Pro Val Thr His Ser Pro Ala Pro Gln Pro Ser TCC ACC TTC GAC A.CC ATG TCG CCG GCG CCT GTC ATC CCC TCC AAC ACC 389 Ser Thr Phe A ~ p Thr Ser Ser Pro Ala Pro Val Pro Island Ser Asn Thr Asp Tyr Pro Gly Pro His His Phe Glu Val Thr Phe Gln Gln Ser Ser ACG GCC A ~ G TCA GCC ACC TGG ACG TAC TCC CGC CTC TTG AAA AAA CTC 485 Thr Ala Ly ~ Ser Ala Thr Trp Thr Tyr Ser Pro Leu Leu Lys Ly ~ Leu Tyr Cys Gln Island Ala Lys Thr Cyq Pro Island Gln Island Lys Val Ser Thr Pro Pro Pro Pro Gly Thr Ala Isle Arg Ala Pro Met Val Tyr Ly- ~ Ly ~

Ala Glu His Val Thr Asp Val Val Ly ~ Arg Cy ~ Pro A- ~ n Hiq Glu Leu GGG AGG GAC TTC AAC GAA GGA. CAG TCT GCT CCA GCC AGC CAC CTC ATC 677 , CA 0224 ~ 242 1998-07-28 WO 97/28186 ~ 2 PCT ~ R97 / 00214 Gly Arg Asp Phe Asn GlU Gly Gln S ~ r Ala Pro Ala Ser ~ is Leu Ile Arg Val Glu Gly Asn Asn Leu Ser Gln Tyr Val A ~ p Asp Pro Val Thr Gly Arg Gln Ser Val Val Val Pro Glu Pro Pro Gln Val Gly Thr Glu Phe Thr Thr Island Leu Tyr Asn Phe Met Cy ~ Asn Ser Ser Cys Val Gly Gly Met Asn Arg Arg Pro Ile Leu Ile Ile Ile Thr Leu Glu Met Arg Asp Gly Gln Val Leu Gly Arg Arg Ser Phe Glu Gly Arg Ile Cys Ala Cys Pro Gly Arg Asp Arg Lys Ala Asp Glu A ~ p His Tyr Arg Glu CAG CAG GCC CTG AAC GAG AGC TCC GCC AAG AAC GGG GCC GCC AGC AAG 1013 .Gln Gln Ala Leu Asn Glu Ser Ser Ala Lys Asn Gly Ala Ala Ser Lys Arg Ala Phe Ly ~ Pro Gln Ser Pro Ala Pro Val Ala Leu Gly Ala Gly Val Lyq Lyq Arg Arg Hiq Gly Asp Glu A ~ p Tyr Tyr Tyr Leu Gln Val CGA GGC CGG GAG AAC TTT GAG ATC CTG ATG AAG CTG AAA GAG AGC CTG llS7 Arg Gly Arg Glu Asn Phe Glu Ile Leu Met Ly_ Leu Lyq Glu Ser Leu Glu Leu Met Glu Leu Val Pro Pro Gln Leu Val A ~ p Ser Tyr Arg Gln 380,385,390 Gln Gln Gln Gln Leu Leu Gl Pro Pro Ser Ser ~ Leu Gln Pro Pro Ser Tyr GGG CCG GTC CTC TCG CCC ATG AAC A ~ G GTG CAC GGG GGC ATG AAC AAG 1301 Gly Pro Val Leu Pro Ser Met Asn Ly ~ Hi Val ~ Gly Gly Met Asn Ly ~

Leu Pro Ser Val Aqn Gln Leu Val Gly Pro Gln Pro Pro Hi- ~ Ser Ser Ala Ala Pro Pro Aqn Leu Gly Pro Pro Gly Pro Gly Met Leu Asn Asn 440,445 450,455 His Gly His Ala Val Pro Ala Asn Gly GlU Ser Ser Ser His Ser Ser Ala Gln Ser Met Ser Ser Gly Ser HiY Pro Cys Pro Pro Pro Tyr WO 97128186 ~ 3 PCT ~ FR97 / 00214 His Ala A ~ p Ser Ser Pro Leu Ser Phe Leu Gly Leu Leu Gly Cys Pro 490 ~ 95 ~ 500 A ~ n Cys Glu Tyr Tyr Phe Thr Ser Gln Gly Leu Gln Ser Tire Tyr His Leu Gln A ~ n Leu Thr Island Glu Asp Leu Gly Ala Leu Lys Island Pro Glu CAG TAC CGC ATG AcC ATC TGG CGG GGC CTG CAG GAC CTG AAG CAG GGC 1685 Gln Tyr Arg Met Thr Ile Trp Arg Gly Leu Asp Gln Leu Lya Gln Gly 540,545,550 CAC GAC TAc AGc ACC GcG CAG CAG CTG CTC CGC TCT AGC AAC GCG GCC 1733 His Asp Tyr Ser the hr Ala Gln Gln Leu Leu Arg Ser Ser A ~ n Ala Ala 555,560,565 ACC ATC TcC ATC GGC GGC TcA GGG GAA CTG CAG CGC CAG CGG GTC ATG 1781 Thr I1e Ser Gly Gly Ser Gly Glu Gln Arg Gln Arg Val Met Glu Ala Val His Phe Arg Arg Arg Hi ~ Thr Thr Thr Island Pro Asn Arg GGC GGC CCA GGC GGC GGC CCT GAC GAG TGG GCG GAC TTC GGC TTc GAC 1877 Gly Gly Pro Gly Gly Gly Pro Asp Aspen Tru Ala Asp Phe Gly Asp Phe Leu Pro A ~ p Cys Ly ~ Ala Arg Ly ~ Gln Pro Ile Lys Glu Glu Phe Thr GAG GCC GAG ATC CAC TGAGGGCCICC GC ~ rGG ~ lGC AGCCTGCGCC ACCGCCCAGA 1980 Glu Ala Glu Hi Island ~

GACCCAAGCT GCCTCCCCTC TCCTTCCTGT GTGTCCAAAA ~ L ~ CCTCAGG AGGCAGGACC 2040 AGGAAAGGCC CA ~ CACCGA AGCC ~ CTGT GGACAGCCTG AGTCACCTGC AGAACC 2156 (2) INFORMATION FOR SEQ ID NO: 6:
ti) SRISTIC CHARACTERISTICS OF THE SEQUENCE:
(A) LON ~ U ~ UK: 636 acid ~ amin ~ s (B) TYPE: amune acid (D) CONFIGURATION:}; n ~ ire (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:
Ala Gln Ser Thr Ala Thr Ser Pro Gly Gly Thr Thr Phe Glu ~ is Leu Trp Ser Ser Glu Pro Leu Asp Ser Thr Asp Asp Tyr Pro Leu 2û 25 3û
Ser Gln Ser Gly Gl Asn Asn Glu Val Gly Val Gly Thr Asp Ser Ser Ser Met Asp Val Phe Hi ~ Leu Glu Gly Met Thr Thr Ser Val Met Ala Gln Phe A ~ n Leu Leu Ser Ser Thr Met A ~ p Gln Met Ser Ser Arg Ala Ala CA 0224 ~ 242 1998-07-28 WO 97/28186 54 PCT ~ FR97 / 00214 ~ er Pro Ala Ser Pro Thr Pro Glu His Ala Pro Ala Ser Pro Thr His ~ Pro Pro Ala Pro Gln Ser Ser Pro Thr Phe Asp Ser Pro Ser Pro Ala 100 105 llQ
Pro Val Pro Pro Asn Thr Asp Asp Pro Pro Gly Pro His His Phe Glu Val Thr Phe Gln Ser Gln Ser Thr Ala Ser Lys Ser Ala Thr Trp Thr Tyr Ser Pro Leu Leu Lys Leu Tyr Lys Cys Gln Ala Lys Thr Cy3 Pro ~ Gln Ile Lys Val Ser Pro Pro Pro Pro Gly Thr Ala Arg Island ~ Met Pro Val Tyr Lily Lys Ala Glu His Asp Val Val Asp Val Val Ly ~

Arg Cy ~ Pro Asn His Glu Leu Gly Arg Asp Phe Asn Glu Gly Gln Ser Ala Pro Ala ser His Leu Isle Arg Val Glu Gly Asn A ~ n Leu Ser Gln Tyr Val To: sp Asp Pro Val Thr Gly Arg Val Gln Val Val Val Pro Tyr ~ Pro Pro Gln Gln Val Gly Thr Glu Phe Thr Thr Island Leu Tyr A-ln Phe ~ and Cys Asn Ser Ser Cy ~ Val Gly Gly Met Asn Arg Arg Pro Ile Leu 260,265,270 Ile Ile Ile Thr Leu Glu Met Arg A- ~ p Gly Gln Val Leu Gly Arg Arg Ser Phe Glu Gly Arg Cy ~ Ala Cys Pro Arg Gly Arg Arg Ly ~ Ala 290,295,300 Asp Glu A ~ p His Tyr Arg Glu Gln Gln Ala Leu A- ~ n Glu Ser Ser Ala ~ y- ~ Asn Gly Ala Ala Ser Lys Arg Ala Phe Ly ~ Pro Gln Ser Pro Ala ~ al Pro Ala Leu Gly Ala Gly Val Lyq Lyq Arg Arg His Gly A ~ p Glu 340,345,350 A ~ p Thr Tyr Tyr Leu Gln Val Arg Gly Arg Glu Asn Phe Glu Ile Leu Met Ly-l Leu Ly ~ Glu Ser Leu Glu Leu Met Glu Leu Val Gln Pro Pro 370,375,380 Leu Val Asp Ser Tyr Arg Gln Gln Gln Gln Leu Leu Gln Arg Pro Ser ~ is Leu Pro Gln Pro Ser Tyr Pro Gly Val Leu Pro Pro Ser Asn Lys ~ al Hi ': G} y Gly Met Asn Lys Leu Pro Ser Val A ~ n Gln Leu Val Gly 420,425,430 Gln Pro Pro Pro His ser Ser Ala Thr Pro Asn Leu Gly Pro Val Gly Pro Gly Met Leu A ~ n A Hi ~ Gly Hi ~ ~ Ala Val Pro Ala A ~ n Gly CA 02245242 l998-07-28 WO 97/28186 PCTrFR97 / 00214 Glu Ser Ser Ser Ser Ser Ser Ala Gln Ser Met val Ser Gly Ser His ~ ys Thr Pro Pro Pro Pro Tyr His Ala Pro Asp Ser Ser Val Ser Phe ~ 85 990 495 ~ had Thr Gly Leu Gly Cy9 Pro Asn Cys Glu Island Tyr Phe Thr Ser Gln Gly Leu Gln Ser Tyr Island Hi ~ Leu Gln Asn Leu Thr Ile Glu A3p Leu Gly Ala Leu Ly3 Pro Island Glu Gln Tyr Arg Arg Thr Thr Arg Arg 530,535,540 Leu Gln Aqp Leu Lys Gln Gly Hi ~ Aqp Ser Ser Thr Ala Gln Gln Leu ~ Ser Arg Ser Ser A.sn Ala Ser Ala Thr Ser Isle Gly Gly Ser Gly Glu ~ had Gln Arg Gln Arg Val Met Glu Ala Val His Arg Arg Arg His Thr Island Thr Island Pro A ~ n Arg Gly Gly Pro Gly Gly Gly Pro A ~ p Glu 595,600,605 Trp Ala Asp Phe Gly Phe A ~ p Leu Pro A ~ p Cys Ly ~ Ala Arg Lys Gln Pro Il.e Lys Glu Glu Phe Thr Glu Ala Glu Hi Island ~

(2) INFORMATION FOR SEQ ID NO: 7:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 2040 pair ~ of baqeY
(B) TYPE: nucleic acid IC) NUMBER OF BRINS: double (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA
(vi) ORIGIN:
(A) ORGANIZATION: Mu ~ mu ~ culu3 (ix) ADDITIONAL CHARACTERI'STIQU2:
(A) NAME ~ KEY: CDS
(B) LOCATION: 124..1890 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7:
TGAL ~ LCC ~ l GTGGCCTGCA GGGGACTGAG CCAGGGAGTA GATGCCCTGA GACCCCAAGG 60 GACACCCAAG GA ~ ACCTTGC l ~ G ~ LLGAG A ~ AGGGATCG ~ C ~ GCCCAAGAGA 120 AGC ATG TGT ATG GGC CCT GTG TAT GAAT TCC TTG GGG CAG GCC CAG TTC 168 Met Cy ~ Met Gly Pro Val Tyr Glu Ser Leu Gly Gln Ala Gln Phe AAT TTG CTC AGC AGr GCC ATG GAC CAG ATG GGC AGC CGT GCG GCC CCG 216 A ~ n Leu Leu Ser Ser Ala Met Asp Gln Met Gly Ser Arg Ala Ala Pro Ala Ser Pro Tyr Th Pro Glu Hi ~ Ala Ala Ser Pro Ala His Ser Ser Pro Tyr Ala Gln Pro Ser Ser Thr Phe A ~ p Thr Met Ser Pro Ala Pro CA 0224 ~ 242 1998-07-28 Pro Island Pro Asn Asp Thr Pro Asp Pro Gly Pro His His Phe Glu Val ACC INCL. CAG AGC TCG AGC ACT GCC AAG TCG GCC ACC TGG ACA TAC TCC 408 Thr Phe Gln Ser Gln Ser Thr Ser Ala Ser Ser Thr Thr Thr Ser Pro Leu Leu Lys Leu Tyr Lys Cys Gln Ala Lys Island Thr Cys Pro Ile Gln Isle Lys Val Ser Pro Pro Pro Pro Gly Thr Ala Island Arg Ala Met Pro Val Tyr Lys Lya Ala His Glu His Val Asp Asp Val Val Lys Arg Cy5 Pro Asn His Glu Leu Gly Arg Asp Asher Phe Glu Gly Gln Ser Ala Pro Ala Ser His Leu Island Arg Val Glu Gly Asn Asn Leu Ala Gln Tyr Val Asp Asp Pro Val Thr Gly Arg Gln Ser Val Val Val Pro Tyr Glu Pro Pro Gln Val Gly Thr Glu Phe Thr Thr Leu Tyr A ~ n Phe Met Cys Asn Ser Ser Cy5 Gly Val Gly Met Asn Arg Arg Pro Ile Leu Val Ile Ile Thr Leu Glu Thr Arg Asp Gly Gln Val Leu Gly Arg Arg Ser Phe Glu Gly Arg Island Cys Ala Cys Pro Gly Arg A p Arg Lys Ala Asp GAA CAT GAC CAT TAC CGG CAA GAG. AGC GCT CTG AAT AG GAA ACC ACC AAA 936 Glu Asp His Tyr Arg Glu Gln Gln Ala Leu Asn Glu Ser Thr Thr Lys 260,265,270 Asn Gly Ala Ala Ser Lya Arg Ala Phe Ly ~ Pro Gln Ser Pro Ala Ile Pro Ala Leu Gly Thr Asn Val Lys Ly ~ Arg Arg Hi ~ Gly Asp Glu A ~ p 290,295,300 Met Phe Tyr Met His Val Arg Gly Arg Glu Aan Phe Glu Ile Leu Met Ly ~ Val Lya Glu Ser Leu G1U Leu Met Glu Leu Val Pro Pro Gln Leu Val Asp Ser Tyr Arg Gln Gln Gln Gln Gln Gln Gln Leu Leu Gln Arg Pro 340,345,350 WO 97/28186 57 PCT ~ FR97 / 00214 AGT CAC CTG CAG CCT CCA TCC TAT GGG cCC GTG CTC TCC CcA ATG AAC 1224 Ser His Gln Pro Pro Ser Ser Gly Pro Pro Leu Leu Ser Pro Asn Lys Val Hls Gly Gly Val Asn Lys Leu Pro Val Val Asn Gln Leu Val 370,375,380 Gly Pro Gln Pro Hi Ser Ser Ala Pro Gly Pro Asn Leu Gly Pro ATG GGC TCC GGG ~ TG CTC AAC AGC CAC GGC CAC AGC ATG CCG GCC AAT 1368 ~ t Gly Ser Gly Met Leu Asn Ser His Gly His Ser Met Pro Ala Asn Gly Glu Met Asn Gly Gly His Ser Ser Gln Thr Met Val Ser Gly Ser ~ 20,425,430 His Cys Pro Thr Pro Pro Pro Tyr Hi ~ Ala Pro Asp Ser Ser Leu Val Ser Phe Leu Thr Gly I.eu Gly Cy ~ Pro A ~ n Cys Glu Island Cy ~ Phe Thr Ser CAA GGG TTG CAG P ~ GC ATC TAC CAC CTG CAG AAC CTT ACC ATC GAG GAC 1560 Gln Gly Leu Gln Ser Tyr Island Hi ~ Leu Gln A ~ n Leu Thr Island Glu Asp Leu Gly Ala Leu Ly3 Pro Val Asp Gln Tyr Arg Met Thr Ile Trp Arg Gly Leu Gln Asp Leu Lys Gln Ser Hi3 A ~ p Cy ~ Gly Gln Leu Leu Leu Ser Ser Asn Ala Ala Thr Ser Ser Gly Gly Ser Gly Glu SlS 520 525 Gln Arg Gln Arg Arg Gla Ala Val Val Eis Phe Arg Arg Arg His Arg 530,535,540 ACC ATC AC ~ ATC CCC AAC CGT GGA GGC GCA GGT GCG GTG ACA GGT CCC 1800 Thr Island Thr Island Pro Asn Arg Gly Gly Ala Gly Ala Val Thr Gly Pro GAC GAG TGG GCG G ~ C TTT GGC TT GAC CTG CCT GAC TGC AAG TCC CGT 1848 Asp Glu Trp Ala A. ~ p Phe Gly Phe A- ~ p Leu Pro Asp Cy Ly ~ Ser Arg Lys Gln Pro Ly Island ~ Glu Glu Phe Thr Thr Glu Thr Glu Ser 5 ~ 0 585 TGAGGA ~ CGT AC ~ l ~ ..l ~ IC ~ L ~ 1CC1LC ~ .Cl ~ l ~ GA AA ~ I ~ l ~ ll GGAAGTGGGA 1950 C ~ l ~ llGG ~ l GTGCCCACAG AAACCAGCAA GGAC ~ l ~ lG CCGGATGCCA TTCCTGAAGG 2010 GAAGTCGCTC ATGAACT.AAC ICC ~ L ~ ~ lGG 2040 (2) INFORMATION FOR SEQ ID NO: 8:
(i) CHARACTERISTICS OF THE SE ~ u ~
(A) LENGTH: 589 acid ~ amin ~

CA 0224 ~ 242 1998-07-28 WO 97128186 ~ 8 PCT ~ R97 / 00214 - (B) TYPE: Amino Aclde ~ D) CONFIGURATION: linealre (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:
Met Cys Met Gly Pro Tyr Tyr Glu Ser Gly Gln Ala Gln Phe Asn Ser Leu Al Ser Ala Met Asp Gln Met Gly Ser Arg Ala Ala Pro Ala Ser Pro Tyr Thr Pro Glu His Ala Ala Ser Ala Pro His Ser Pro Pro Tyr Ala Gln Ser Pro Ser Ser Thr Phe Asp Thr Ser Ser Ser Pro Ala Pro Val Pro Pro Asn Thr Asp Pro Tyr Pro Gly Pro His His Phe Glu Val Thr Phe Gln Ser Gln Ser Thr Ala Ser Ser Ser Thr Trp Ser Ser Pro Leu Leu Lys Leu Tyr Lys Cys Gln Ala Lys Island Thr Cys Pro Gln Island Ile Lys Val Ser Pro Pro Pro Pro Gly Thr Ala Arg Ala Met Island Pro Val Tyr Ly ~ Ly- ~ Ala Glu His Val Thr Asp Island Val I ys Arg Cys Pro A: sn Hi Glu Leu Gly Arg A ~ p Phe Asn Glu Gly Gln Ser Ala Pro Ala Ser Hi ~ l Leu Island Arg Val Glu Gly Asn Asn Leu Ala Gln Tyr Val 165 170. 175 Asp Asp Pro Val Thr Gly Arg Gln Ser Val Val Pro Val Tyr Glu Pro Pro Gln Val Gly Thr Glu Phe Thr Thr Leu Tyr Asn Phe Met Cys Asn Ser Ser Cys Val Gly Gly Met Asn Arg Arg Pro Ile Leu Val Ile Ile Thr Leu Glu Thr Arg A ~ p Gly Gln Val Leu Gly Arg Arg Ser Phe Glu Gly Arg Island Cys Ala Cy: ~ Pro Gly Arg A ~ p Arg Lys Ala A ~ p Glu Asp His Tyr Arg Glu Gln Gln Ala Leu A3n Thr Glu Thr Thr Ly ~ A ~ n 260,265,270 Gly Ala Ala Ser Ly Arg Ala Phe Ly ~ Pro Gln Ser Pro Pro Ala Island Ala Leu Gly Thr A ~ n Val Ly ~ Ly ~ Arg Arg His Gly A ~ p Glu Asp Met 290,295,300 Phe Tyr Met Hi3 Val Arg Gly Arg Glu A- ~ n Phe Glu Ile Leu Met Ly ~

Val Ly ~ Glu Ser Leu Glu Leu Met Glu Leu Val Gln Pro Pro Leu Val A ~ p Ser Tyr Arg Gln Gln Gln Gln Gln Gln Leu Leu Gln Arg Pro Ser Ser 340,345,350 CA 02245242 l998-07-28 WO 97/28186 S9 PCT ~ FR97 / 00214 His Leu Gln Pro Ser Ser Pro Gly Val Leu Pro Ser Ser Met Asn Lys Val His Gly Gly Val Asn Ly ~ Leu Pro Ser Val Acn Gln Leu Val Gly 370,375,380 Gl ~ Pro Pro Hi Pro Ser Ser Ala Pro Gly Pro Asn Leu Gly Pro Met ~ ly Ser Gly Met Leu Ser Asn Ser Hi3 Gly His Ser Pro Met Ala Asn Gly ~ 05 410 415 ~ Read Met Asn Gly Gly His Ser Ser Gln Thr Met Val Ser Gly Ser H
420,425,430 Cy3 Pro Pro Thr Pro Pro Hi ~ Ala Asp Ser Pro Ser Val Ser Phe Leu Thr Gly Leu Gly Cy-Y Pro Asn Cy ~ Ile Glu Cy3 Phe Thr Ser Gln Gly Leu Gln Ser Tyr Island His Leu Gln AYn Leu Thr Ile Glu Asp Leu ~ ly Ala Leu LyY Val Pro A ~ p Gln Tyr Arg Met Thr Ile Trp Arg Gly 4 ~ 5 490 49S
Gln A3p Gln Ser Hi3 A3p Cy-Y Gly Gln Gln Leu Leu Arg Ser Ser Ser Asn Al.a Ala Thr Ile Ser Gly Gly Ser Gly Glu Leu Gln Arg Gln Arg Va.l Met Glu Ala Val Hi3 Phe Arg Arg Arg Hi ~ Thr 530,535,540 Thr Island Pro Island Asn Arg Gly Gly Ala Gly Ala Val Thr Gly Pro A ~ p Glu Trp Ala A ~ p Phe Gly Phe A ~ p Leu Pro Cy3 Ly Ly ~ Ser Arg LyY

Gln Pro Island Ly3 Glu Glu Phe Thr Glu Glu Glu Ser Hi ~
580,595 (2) INFORMATION FOR SEQ ID NO: 9:
(i) CHARACTERISTICS OF THE SEQUENCE:
AI LON ~ U ~ UK: 758 pair3 of baYe ~
~ B TYPE: nucleic acid C ~ NUMBER OF BRINS: double : D, CONFIGURATION l; n ~ aire (ii) TYPE OF MOLECULES: cDNA
(vi) ORIGIN:
(A) ORG ~ ISME: mu3 mu ~ culus (ix) ADDITIONAL CHARACTERISTIC:
~ A) NAME / KEY: CDS
(B) LOCATION: 389,757 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9:

CCGCTGGGGC TA ~ LLG ~ GCG ACGCGCGCCA AGC ~ CG6CG GGAAGGAGGC GGGAGGAGCG 120 GGGCCCGAGA CCCCGACTCG GGCAGAGCCA G ~ GG & CAGG CGGGGCGCGC GTGGGAGCCA 180 CA 0224 ~ 242 1998-07-28 WO 97t28186 60 PCTAFR97 / 00214 Met Ser Gly Ser Val Gly Glu Met Ala Gln Thr Ser Ser Ser Ser Ser Ser Ser Thr Phe Glu His Leu Trp Ser Ser Leu Glu Pro Ser Asp Asp Thr Tyr Phe Asp Pro Leu Gln Pro Ser Gln Gly Thr Ser Glu Ala Ser Gly ser Glu Glu Asn Ser Met Asp Val Phe His Leu Gln Gly Met Ala Gln Phe A ~ n Leu Ser Ser Ser Ala Met Asp Gln Met Gly Ser Arg Ala Pro Ala Pro Pro Tyr Thr Pro Glu His Ala Ala Ser Pro Ala Pro His Ser Pro Pro Ala Pro Ser Ser Pro Phe Asp Thr Met Ser Pro Ala Pro Val Pro Island Ser Asn Thr A ~ p Tyr Pro Gly Pro ~ 2) INFORMATION FOR SEQ ID NO: 10:
~ i) CHARACTERISTICS OF THE SEQUENCE:
(A) L ~ N ~ u ~: uK: 123 acid ~ amine ~
(B) TYPE: amino acid (D) CONFIGURATION: linear ~ ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10:
Ser Ser Gly Ser Gly Glu Ser Met Ala Gln Ser Ser Ser Ser ser Ser ~ er Thr Phe Glu His Leu Trp ser Ser Glu Pro A3p ser Ser Thr Tyr Phe Asp Leu Pro Gln ~ ro Ser Gln Gly Thr Ser Glu Ala Ser Gly Ser Glu Glu Ser A ~ n Met Asp Val Phe His Leu Gln Gly Met Ala Gln Phe A ~ n Leu Leu Ser Ser Ala Met Asp Gln Met Gly Ser Arg Ala Ala Pro Ala Ser Pro Pro Thr Thru Hi Glu ~ Ala Ser Pro Ala Pro Thr Hi ~ Ser 9, 95 WO 97/28186 61 PCT ~ FRg7 / 00214 Pro Tyr Ala Gln Ser Pro Ser Thr Threat Phe Thr Met Ser Pro Ala Pro Val I ~ Pro Ser Asn Thr Acp Tyr Pro Gly Pro (2) INFORMATION FROM SEQ ID NO: 11:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) L ~ U ~ UK: 559 pair ~ of ba3es (B) TYPE: nucleic acid (C) NOY ~ BRE OF BRINS: double (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA
(vi) ORIGIN:
(A ~ ORGANISM: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11:

GGACGTCTTC CACCTGGAGG GCATGACTAC ATCTGTCATG CATCCTCGGC TCCTGCCTcA 120 CCCCTCGGGC CGCCCA ~, ATC CATGCCTCGT CCCACGGGAC ACCAGTTCCC TGGCGTGTGC 240 AGACCCCCCG GCGCCT ~ CCA ~ L ~ ACGT CGGTGACCCC GCACGGCACC TCGCCAcGGC 300 CCAGTTCAAT CTGCTGAGCA GCACCATGGA CCAGATGAGC AGCCGCGCGG CCTCGGCCAG, 360 CTCCACCTTC GACACC ~ TGT CGCCGGCGCC TGTCATCCCC TCCAACACCG ACTACCCCGG 480 GTACTCCCCG cT ~~ AG 559 (2) INFORMATION FOR SEQ ID NO: 12:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A: ~ INFRUITER: 1764 pair ~ of ba ~ e ~
(B ~ TYPE :: Nuclear acid (CI NOME ~ RE OF BRINS: double (D, CONFIGURATION: lin ~ Aire (ii) TYPE OF MOLECULE: cDNA
(vi) ORIGIN:
(A) ORGPNISM: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12:

ATGCTGTACG TCGGTGA.CCC CGCACGGCAC CTCGCCACGG CCCAGTTCAA T ~ T ~ cl ~ AGC 60 AGCACCATGG ACCAGATGAG CAGCCGCr, CrJ GCCTCGGCCA GCCCCTACAC CCCAGAGCAC 120 ACTTTCCAGC AGTCCAGCAC GGCCAAGTCA GCCACCTGGA CGTACTCCCC Gol ~ l ~ AAG 300 CCCCCAGGCA CTGCCAT ~ CG GGCCATGCCT GTTTACAAGA AAGCGGAGCA CGTGACCGAC 420 CA 0224 ~ 242 1998-07-28 WO 97 / Z8186 62 PCT ~ R97 / 00214 GTCGTGAAAC GCTGCCCCAA CCACGAGCTC ÇGGAGGGACT TCAACGAAGG ACAGTCTGCT 480 TTCAAGCAGA GCCCCCCTGC CGTCCCCGCC ~ lLG ~ lGCCG GTGTGAAGAA GCGGCGGCAT 900 GACCCCAGCC TCGTCAGTTT TTTAACAGGA TTGGGGTGTC CA ~ ACTGCAT CGAGTATTTC 1380 GCCCTGAAGA TCCCCGAGCA GTACCGCATG ACCATCTGGC GG ~ C ~ GCA GGACCTGAAG 1500 TCCATCGGCG GCTCAGGGGA ACTGCAGCGC CAGCGG ~ l ~ A TGGAGGCCGT GCACTTCCGC 1620 GTGCGCCACA CCATCACCAT CCCCAACCGC GGCGGCCCAG GC ~ CCC TGACGAGTGG 1680 (2) INFORMATION FOR SEQ ID NO: 13:
(i) CHARACTERISTICS OF THE SEQUENCE:
~ A) L ~ N ~ u ~ UK: 587 acid ~ friend ~ és (B ~ TYPE: amino acid (D) CONFIGURATION: 1 day (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13:
Met Leu Tyr Val Gly A ~ Pro p Ala Arg Hi ~ Ala Thr Ala Al Gln Phe l 5 10 15 A ~ n Leu Leu Ser Ser Ser Met Asp Gln Ser Ser Ser Arg Ala Ala Ser Ala Ser Pro Pro Thr Thru Glu Hi ~ Pro Ala Ala Ser Pro Thr ~ is Ser Pro Tyr Ala Gln Ser Pro Ser Thr Threat Phe Thr Met Ser Pro Ala Pro WO 97 / Z8186 63 PCT ~ R97 / 00214 Pro Island Pro Asn Asp Thr Pro Asp Pro Gly Pro His His Phe Glu Val Th ~ Phe Gln Ser Gln Ser Thr Ala Ser Lys Ser Thr Ala Thr Trp Thr Ser Ser Pro Leu Leu Lys Leu Tyr Lys Cys Gln Ala Lys Island Thr Cys Pro Ile Gln Isle Lys Val Ser Pro Pro Pro Pro Gly Thr Ala Island Arg Ala Met Pro Val Tyr Lily Lys Ala Glu His Val Thr Asp Val Val Lys Arg Cys Pro Asn His Glu Leu Gly Arg Asp Phe Asn Glu Gly Gln Ser Ala Pro Ala Ser His Leu Arg Island G1U Gly Asn A ~ n Leu Ser Gln Tyr Val Asp Asp Pro Val Thr Gly Arg Gln Ser Val Val Val Pro Tyr Glu Pro Pro Gln Val Gly Thr Glu Phe Thr Thr Leu Tyr Asn Phe Met Cys Asn Ser Ser Cy Val Gly Gly Met Asn A ~ g Arg Pro Ile Leu Ile Ile Ile Thr Leu Glu Met Arg Asp Gly Gln Val Leu Gly Arg Arg Ser Phe Glu Gly Arg Cys Ala Cys Pro Gly Arg Asp Asp Arg Lys Ala Asp Glu A- ~ p His ryr Arg Glu Gln Gln Ala Leu Asn Glu Ser Ser Ala Lys 260,265,270 Asn Gly Ala Ala Ser Lys Arg Ala Phe Lys Pro Gln Ser Pro Ala Val Pro Ala Leu Gly Ala Gly Val Ly ~ Arg Arg Lips Its Gly Asp Asp Asp 290,295,300 Thr Tyr Tyr Leu Gln Val Arg Gly Arg Glu Asn Phe Glu Ile Leu ~ and Lys Leu Lys (~ lu Leu Leu Leu Leu Glu Leu Leuk Pro Gln Pro Leu Leu Val A ~ p Ser ~ yr Arg Gln Gln Gln Gln Leu Leu Gln Arg Pro Ser His '~ 40 345 350 Leu Gln Pro Pro Ser Tyr Pro Gly Val Leu Ser Pro Pro Asn Lys Val His Gly Gly ~ let A ~ n Lys Leu Pro Ser Val A ~ n Gln Leu Val Gly Gln 370,375,380 Pro Pro Pro EIi Ser Ser Ala Ala Pro Thr: Leu Gly Pro Gly Pro Pro Gly Met Leu Asn A ~ n His Gly Hi ~ Ala Val Pro Ala Asn Gly Glu Ser Ser Ser Hi ~ Ser Ala Gln Ser Ser Val Ser Gly Ser Hi ~ Cys 420,425,430 Thr Pro Pro Pro Hi Pro ~ Ala A- ~ Pro Ser Pro Leu Val Ser Phe Leu Thr Gly Leu Gly Cys Pro Asn cy5 Glu Island Tyr Phe Thr Ser Gln Gly Leu Gln Ser Tyr Island His Leu Gln Asn Leu Thr Ile Glu Asp Leu Gly Ala Leu Lys Pro Island Glu Gln Tyr Arg Met Thr Ile Trp Arg Gly Leu Gln Asp Leu Lys Gln Gly His Asp Asp Ser Ser Thr Ala Gln Gln Leu Leu Arg Ser Ser Asn Ala Ala Thr Ile Ser Gly Gly Ser Gly Glu Leu Gln Arg Gln Arg Val Met Glu Ala Val His Phe Arg Val Arg Hls Thr 530,535,540 Isle Thr Island Pro Asn Arg Gly Gly Pro Gly Gly Pro Gly Asp Trp Ala Asp Phe Gly Phe Asp Leu Pro A ~ p Ly Cys ~ Ala Arg Lys Gln Pro Ly Island ~ Glu Glu Phe Thr Ala Glu Glu Island His 580,585 (2) INFORMATION FOR SEQ ID NO: 14:
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 1521 pairq of ba eq (B) Nucleic acid type (C) NUMBER OF BRINS: double (D) CONFIGURATION: lin ~ aire (ii) TYPE OF MOLECULE: cDNA
(vi) ORIGIN:
(A) ORGANIZATION: Homo qapienq (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19:
ATGCTGTACG TCGGTGACCC CGCACGGCAC CTCGCCACGG CCCAGTTCAA ~ LG ~ G ~ GC 60 A ~ LIC ~ AGC AGTCCAGCAC GGCCAAGTCA GCCACCTGGA CGTACTCCCC G ~ IL ~ AAG 300 GTCGTGAAAC GCTGCCCCAA CCACGAGCTC GGGAGGGACT TCAACGAAGG ACA ~ L ~ T ~ 480 GTCACCGGCA GGCAGAGCGT C ~ LG ~ LGCCC TATGAGCCAC CACAGGTGGG GACGGAATTC 600 TTTGAGGGCC GCA. ~. '~ CGC CTGTCCTGGC CGCGACCGAA AAGCTGATGA GGACCACTAC 780 TTCAAGCAGA GCCCCCCTGC CGTCCCCGCC CLL ~ l ~ CCG GTGTGAAGAA GCGGCGGCAT 900 WO 97/28186 65 PCT ~ 97/00214 GGAGACGAGG ACAC & TACTA CCTTCA ~ GTG CGAGGCCGGG AGAACTTTGA GATCCTGATG 960 A ~ GCTGAAAG AGAGCCTGGA GCTGATGGAG TTGGTGCCGC AGCCACTGGT GGACTCCTAT 1020 r AGTGCCAGCC AACGGCGAGA TGAGCAGCAG CCACAGCGCC CAGTCCATGG TCTCGGGGTC 1140 (2) INFORMATION FOR SEQ ID NO: 1S:
(i) CHARACTERISTIC OF THE SEQUENCE:
(A) LO ~ rGUEUR: 506 amino acid ~
(B) TYE'E: amino acid (D) COWFIGURATION: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15:
Met Leu Tyr Val Gly A ~ p Pro Ala Arg His Leu Ala Thr Ala Gln Phe A ~ n Leu Leu Ser Ser Ser Met Asp Gln Ser Ser Ser Arg Ala Ala Ser Ala Ser Pro Pro Thr Thru Glu Hi ~ Ala Pro Sera Pro Thr Hi ~ Ser Pro Tyr Ala Gln Ser Pro Ser Thr Phe Ser A3p Ser Pro Pro Ala Pro Val Ile Pro Ser A ~ n ~ hr A ~ p Pro Tyr Pro Gly His His Phe Glu Val Thr Phe Gln Ser Gln Ser Ser Ala Ly Ser Ser Ser Thr Thr Thr Ser Ser Pro Leu Leu Ly ~ Leu Tyr Ly Cy ~ Gln Ile Ala Ly ~ Thr Cys Pro Ile Gln Island Ly ~ Pro Ser Pro Pro Pro Gly Thr Ala Arg Ala Island Pro Met Pro Val Tyr Ly Lys Ly Ala Glu Hi ~ Asp Val Asp Asp Val Val Lys Arg Cy3 Pro Asn Hi ~ Glu Leu & ly Arg A ~ p Phe Asn Glu Gly Gln Ser Ala Pro Ala Ser ~ is Leu Arg Island Glu Gly A n A ~ n Leu Ser Gln Tyr Val A ~ p To Pro Val Thr Gly Arg Gln Ser Val Val Val Pro Tyr Glu CA 02245242 l998-07-28 WO 97/28186 66 PCT / F ~ 97/00214 Pro Gln Gln Val Gly Thr Thru P Thr Thr Leu Leu Asn Phe Met Cys A ~ n Ser Ser Cys Val Gly Gly ~ and Asn Arg Arg Pro Ile Leu Ile Ile Ile Thr Leu Glu Met Arg Asp Gly Gln Val Leu Gly Arg Arg Ser Phe Glu Gly Arg Cys Ala Cys Pro Gly Arg Asp Asp Arg Lys Ala Asp Glu A ~ p Hls Tyr Arg Glu Gln Gln Ala Leu Asn Glu Ser ser Ala Lys 260,265,270 Asn Gly Ala Ala Ser Lys Arg Ala Phe Lys Pro Gln Ser Pro Ala Val Pro Ala Leu Gly Ala Gly Val Ly ~ Lyq Arg Arg His Gly Asp Glu Asp 290,295,300 Thr Tyr Tyr Leu Gln Arg Val Gly Arg Glu Asn Phe Glu Ile Leu Met Lys Leu Lys Leuk Glu Ser Leu Leu Leuk Glu Met Leu Leu Pro Gln Pro Leu Val Aap Ser Tyr Arg Gln Gln Gln Gln Leu Leu Gln Arg Pro Pro Arg.
340,345,350 Asp Ala Gln Pro Gln Pro Trp Arg Ser Ala ser Gln Ar ~ Arg Asp Glu Gln Gln Pro Gln Arg Pro Val Hi ~ Gly Leu Gly Val Pro Leu Hi ~ Ser 370,375,380 Ala Thr Pro Leu Pro Arg Arg Pro Gln Pro Arg Asp Gln Leu Gly Ala Leu Lys Pro Island Glu Gln Tyr Arg Met Thr Ile Trp Arg Gly Leu Gln Aqp Leu Lys Gln Gly His Asp Asp Ser Ser Thr Ala Gln Gln Leu Leu Arg 420,425,430 Ser Ser A ~ n Ala Ala Thr Ile Ser Ile Gly Gly Ser Gly Glu Leu Gln Arg Gln Arg Val Met Glu Ala Val Hi ~ Phe Arg Arg Arg Hiq Thr Ile Thr Island Pro A ~ n Arg Gly Gly Pro Gly Gly Gly Pro Asp Glu Trp Ala Asp Phe Gly Phe A p Leu Pro A ~ p Cy ~ Lys Ala Arg Ly ~ Gln Pro Ile Lys Glu Glu Phe Thr Glu Ala Glu Island His 500,505 (2) INFORMATION FOR SEQ ID NO: 16:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A ~ L ~ N ~ u ~ uK: 1870 pairY of baqe ~
(B TYPE: nucleic acid (C NUMBER OF LRINS: double (Dl CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA
(vi) ORIGIN:
(A) ORGANIZATION: Homo sapienq =

CA 0224 ~ 242 l998-07-28 WO 97/28186 67 PCT ~ FR97 / 00214 .
~ (ix) ADDITIONAL CHARACTERISTIC:
~ A) NAME / KEY: CDS
(~) LOCATION: 104..1867 r (Xl) DESC ~ IPTION OF THE SEQUENCE: S ~ Q ID NO: 16:

AAGCcGGGGG AATAATGAGG TGGTGGGCGG AAcGGATTCC AGC ATG GAC GTC TTC 115 Met Asp Val Phe CAC CTG GAG GGC ATG AcT ACA TCT GTC ATG Gcc CAG TTC AAT cTG CTG 163 Hls Leu Glu Gly Met Thr Thr Ser Val Met Ala Gln Phe Asn Leu Leu AGC AGC ACC ATG GAC CAG ATG AGC AGC CGC GCG GCC TCG GCC AGC CcC 211 Ser Ser Thr Met Asp Gln Ser Ser Ser Ala Ser Ser Ala Ser Pro Tyr Thr Pro Glu Hi ~ Ala Ala Ser Pro Val Thr His Ser Pro Tyr Ala Gln Pro Ser Ser Thr Threat Phe Thr Met Ser Pro Ala Pro Pro Val Pro Ser Asn Thr A- ~ p Tyr Pro Gly Pro Hi Hi ~ Phe Glu Val Thr Phe Gln Gln Ser Ser Thr ~ Lys Ser Ala Thr Trp Thr Tyr Ser Pro Leu Leu Ly ~ Lys Leu Tyr Cys Gln Ile Ala Ly ~ Thr Cy ~ Pro Ile Gln Ile Ly ~
~ 05 110 115 Val Ser Thr Pro Pro Pro Gly Thr Ala Isle Arg Ala Met Pro Val Tyr Ly ~ Ly Ala Glu His Val Asp Asp Val Val Ly ~ Arg Cyq Pro Asn CAC GAG CTC GGG ~ GG GAC TTC GGA AAC GGA CAG TCT GCT CCA GCC AGC 595 Hi ~ Glu Leu Gly Arg AYP Phe Aqn Glu Gly Gln Ser Ala Pro Ala Ser 150 lSS 160 His Leu Isle Arg Val Glu Gly A ~ n Asn Leu Ser Gln Tyr Val Acp A ~ p Pro Val Thr Gly ~ rg Gln Ser Val Val Pro Val Pro Glu Pro Gln Gln 185 lg0 195 Val Gly Thr Glu Phe Thr Thr Ile Leu Tyr A ~ n Phe Met Cy ~ A ~ n Ser Ser Cys Val Gly Gly Met A ~ n Arg Arg Pro Ile Leu Ile Ile Ile Thr ~ 215 220 225 Leu Glu Met Arg A ~ p Gly Gln Val Leu Gly Arg Arg Ser Phe Glu Gly CA 0224 ~ 242 1998-07-28 WO 97/28186 68 PCTA ~ R97 / 00214 230 23s 2qo CGC ATC TGC GCC TGT CCT GGC CGC GAC CGA A ~ GCT GAT GAG GAC CAC 883 Arg Cy3 Ala Cys Pro Gly Arg Asp Asp Arg Lys Ala Asp Glu Asp Asp Tyr Arg Glu Gln Al Gln Gln Asn Glu Ser Ser Ala Lyq Asn Gly Ala Ala Ser Lys Arg Ala Phe Lys Pro Gln Ser Pro Ala Pro Pro Ala Leu 280,285,290 Gly Ala Gly Val Lys Lys Arg Arg His Gly Asp Asp Glu Asp Tyr Tyr CTT CAG GTG CGA GGC CGG GAG AAC TTT GAG ATC CTG ATG AAG CTG A ~ A 1075 Leu Gln Val Arg Gly Arg Glu Asn Phe Glu Ile Leu Met Lys Leu Lys 310,315,320 Glu Ser Leu Glu Leu Met Glu Leu Val Pro Pro Gln Leu Val Asp Ser 325 330 33s 340 Tyr Arg Gln Gln Gln Gln Gln Leu Leu Gln Arg Ser Ser Pro Gln Pro Pro Ser Tyr Gly Pro Pro Val Leu Ser Met A ~ n Lya Val His Gly Gly Asn Lys Leu Pro Ser Val A ~ n Gln Leu Val Pro Gly Gln Pro Pro 375,380,385 Hi ~ Ser Ser Ala Pro Ala Thr Pro Leu Gly Pro Pro Gly Pro Gly Pro Leu Asn Asn Hi ~ Gly Hi ~ Ala Val Pro Ala A3n Gly Glu Ser Ser Ser Ser Hi ~ Ser Ala Gln Ser Met Ser Val & Ser Ser Cys Thr Pro Pro Pro Pro Tyr His Ala A ~ p Pro Ser Leu Val Ser Phe Leu Thr Gly Leu Gly Cys Pro A ~ n Cy3 Ile GlU Tyr Phe Thr Ser Gly Gln Leu Gln Ser Ile Tyr Hi Leu Gln A n Leu Thr Ile Glu Asp Leu Gly Ala Leu Lyq Ile Pro Glu Gln Tyr Arg Met Thr Ile Trp Arg Gly Leu Gln Asp Leu 485,490,495,500 Lys Gln Gly His Asp Ser Ser Tyr Ala Gln Gln Leu Leu Arg Ser Ser Ser Ala Ala Thr Island Ser Gly Gly Ser Gly Glu Leu Gln Arg Gln CA 02245242 l998-07-28 520,525,530 Arg Met Met Glu Ala Val His Arg Phe Arg Arg Hls Thr Ile Thr Ile 535,540,545 Pro Asn Arg Gly Pro Gly Gly Pro Gly Pro Asp Ala Asp Phe GGC TTC GAC CTG CCC GAC TGC AAG CGC CGC AAG CAG CCC ATC AAG GAG 18 ~ 3 Gly Phe A ~ p Leu Pro Asp Cys Lys Ala Arg Lys Gln Pro Ile Lys Glu 565,570,575,580 GAG VAT included AcG GAG GCC GAG ATC CAC TGA 1870 Glu Phe Thr Ala Glu Glu Island His (2) Irrformation for SEQ ID NO: 17:
(i) CHARACTERISTICS OF THE S ~ QU ~ NCL:
(A) LENGTH: 588 aclde ~ amin ~
(B) TYPE: Amino acid (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17:
Met Asp Val Phe Hi ~ Leu Glu Gly Met Thr Thr Ser Val Met Ala Gln l 5 10 15 ~ he Asn Leu Leu Ser Ser Ser Met A p Gln Ser Ser Ser Arg Ala Ala Ser Ala Pro Ser Pro Thr Thru Hi Glu ~ Pro Ala Ser Pro Vala Thr Hls Ser Pro Tire Ala Gl.n Pro Ser Ser Thr Phe A ~ p Thr Met Ser Pro Ala Pro Val Pro Island S ~ r A ~ n Asp Asp Tyr Pro Gly Pro ~ al Thr Phe Gln Gln Ser Ser Thr Ala Lys Ser Ala Thr Trp Thr Tyr Pro Leu Leu L Ly Ly Leu Cy Leune Gln Ile Ala Lyq Thr Cy ~ Pro Ile Gln Ile Ly ~ Val Ser Pro Pro Pro Pro Gly Thr Ala Arg Island l1S 120 125 Ala Met Pro Val Tyr Lily Lys Ala Glu His Val Thr A ~ p Val Val Lys Arg Cys Pro A ~ n Hi ~ Glu Leu Gly Arg A ~ p Phe Asn Glu Gly Gln Ser 145 150 lSS 160 ~ Pro Ala Ser His Leu Isle Arg Val Glu Gly A ~ n A ~ n Leu Ser Gln ~ yr Val A-cp A Pro Val Thr Gly Arg Gln Ser Val Val Val Pro Tyr 180 lbs 190 Glu Pro Pro Gln Va.l Gly Thr Glu Phe Thr Thr Leu Tyr Asn Phe l9S 200 205 Met Cys Asn Ser Ser Cy ~ Val Gly Gly Met A ~ n Arg Arg Pro Ile Leu -CA 0224 ~ 242 l998-07-28 WO 97/28186 PCTn ~ R97 / 00214 Ile Ile Ile Thr Leu Glu Met Arg A ~ p Gly Gln Val Leu Gly Arg Arg 22s 230s 23s 240s Ser Phe Glu Gly Arg Cys Ala Cys Pro Gly Arg Arg Arg Ala Ala 245 2s0 255 ~ sp Glu Asp His Tyr Arg Glu Gln Gln Ala Leu Asn Glu Ser Ser Ala 260,265,270 Lys Asn Gly Ala Ala Ser Lys Arg Ala Phe Lilies Gln Ser Pro Pro Ala Val Pro Ala Leu Gly Ala Gly Val Lys Lys Arg Arg His Gly Asp Glu 290,295,300 Asp Thr Tyr Tyr Leu Gln Val Arg Gly Arg Glu Asn Phe Glu Ile Leu Met Ly ~ Leu Lys Glu Ser Glu Leu Leu Glu Leu Gln Pro Gln Pro 325 330 33s ~ had Val Asp Ser Tyr Arg Gln Gln Gln Gln Leu Leu Gln Arg Pro Ser Ser 340,345,350 His Leu Pro Gln Pro Ser Tyr Pro Gly Pro Leu Ser Pro Met Asn Lys Val Hi ~ Gly Gly Met A ~ n Lys Leu Pro Ser Val A ~ n Gln Leu Val Gly 370,375,380 Gln Pro Pro Pro Ser Ser Ala Ala Pro Asn Pro Leu Gly Pro Pro Gly Pro Gly Met Leu Asn A His Gly His Ala Val Pro Ala A ~ n Gly ~ Read Ser Ser Ser Ser His Ser Ala Gln Ser Ser Val Ser Gly Ser His 420,425,430 Cys Thr Pro Pro Pro Pro Tyr His Ala Asp Ser Ser Ser Val Ser Phe Leu Thr Gly Leu Gly Cy ~ Pro Asn Cys Glu Island Tyr Phe Thr Ser Gln q50 455 460 Gly Leu Gln Ser Tyr Island Hi ~ Leu Gln Asn Leu Thr Ile Glu Asp Leu Gly Ala Leu Lys Pro Island Glu Gln Tyr Arg Met Thr Isle Trp Arg Gly ~ Gln A p Leuyl Gln Gly Hi ~ A- ~ p Ser Ser Thr Gln Gln Gln Leu Ser Ser Ser Asn Ala ~ Ser Ser Island Ser Gly Gly Ser Gly Glu Gln Arg Gln Arg Gln Arg Gla Ala Val His Arg Arg 530,535,540 Thr Island Thr Island Pro Asn Arg Gly Gly Pro Gly Gly Pro Gly Asp Glu 54s 550 555 560 Trp Ala Asp Phe Gly Phe Asp Leu Pro Asp Cys Lily Ala Arg Lys Gln Pro Ile Lys Glu Glu Phe Thr Glu ~ GlU Island His 580,585 (2) IN2'0RMATION FOR S5Q ID NO: 18:
(i) CHARACTERISTICS OF THE SEQUENCE:

CA 0224 ~ 242 1998-07-28 (A) LENGTH 1817 pair of baqes TYPE: nucleic acid (C) NUMBER OF DOUBLE BRINS
(D) linear CONFIGURATION
(ii) TYPE OF MOLECULE cDNA
(vi) ORIGIN:
(A) ORGJANISM Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18 CAACCCAGCT CCACCI'TCGA CACCATGTCG CCGGCGCCTG TCATCCCCTC CAACACCGAC 360 ACCTGGACGT ACTCCCCGCT CTTGAAGAAA CTCTAcTGCC AGATCGCCAA GACATGCCCC 480 TACAAGA ~ AG CGGAGCACGT GACCGACGTC GTGAAACGCT GCCCCAACCA CGAGCTCGGG 600 AGGGACTTCA ACGAAGGACA ~ L ~ G ~ lCCA GCCAGCCACC TCATCCGCGT GGAAGGCAAT 660 AAGAACGGGG CCGCCAGCAA GC ~ .GC ~ .lC AAGCAGAGCC CCCCTGCCGT CCCCGCCCTT 1020 GGTGCCGGTG TGAAGA ~ GCG GCGGCATGGA GACGAGGACA CGTACTACCT TCAGGTGCGA 1080 GGCCGGGAGAGA ACTTTG ~ GAT CCTGATGAAG CTGAAGAGA GC ~ lG ~ AGCT GATGGAGTTG 1140 GTGCCGCAGC CA ~ L ~ rG ~ A CTCCTATCGG CAGCAGCAGC AG ~ CACA GAGGCCGAGT 1200 ATGAACAAGC TGCCCTCCGT CA ~ CCAGCTG GTGGGCCAGC CTCCCCCGCA CAGTTCGGCA 1320 GCTACACCCA AC ~ lGGGGCC C ~ l ~ & GCCCC GGGATGCTCA ACAACCATGG CCACGCAGTG 1380 CCAGCCAACG GCGAGA'rGAG CAGCAGCCAC AGCGCCCAGT CCALG ~ .C GGGGTCCCAC 1440 AGATCCCCGA GCAGTACCGC ATGACCATCT GGCG ~ GGC ~ l GCAGGACCTG AAGCAGGGCC 1560 CA 0224 ~ 242 1998-07-28 WO 97/2818 ~ 72 PCT ~ FR97 / 00214 ~ AGGCCGAGAT CCACTGA 1817 (2) INFORMATION FOR SEQ ID NO: 19:
(1) CHARACTERISTICS ~ S ~ QUENC ~:
(A) LENGTH: 499 acid ~ amlnated (B) TYPE: amino acid (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: protelne (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19:
Ala Gln Ser Thr Ala Thr Ser Pro Gly Gly Thr Thr Phe Glu His Leu Ser Trp Ser Ser Glu Pro Asp Asp Ser Asp Tyr Asp Asp Pro Phe Ser Gln Ser Gly Gl Asn Asn Glu Val Gly Val Gly Thr Asp Ser Ser Ser Met Asp Val Phe His Leu Glu Gly Met Thr Thr Ser Val Met Ala Gln Phe Asn Leu Ser Leu Ser Ser Met Asp Gln Ser Ser Ser Arg Ala ALa Ser Ala Ser Pro Tyr Thr Pro Glu His Ala Ala Ser Pro Val Thr His Ser Pro Ala Gln Pro Ser Ser Ser Thr Phe Thr Thr Met Ser Ser Pro Ala Pro Pro Island Pro Ser Asn Thr A ~ p Pro Tyr Pro Gly His Hls Phe Glu Val Thr Phe Gln Gln ser Ser Thr Ala Ly ~ Ser Ala Thr Trp Thr Tyr Ser Pro Leu Leu Ly ~ Lyq Leu Tyr CyY Gln Ile Ala Ly ~ Thr Cys Pro lg5 150 155 160 Ile Gln Ile Lys Val Ser Pro Pro Pro Pro Gly Thr Ala Arg Island Ala Met Pro Val Tyr Lys Lyq Ala Hi Glu Val Thr Asp Val Val Lys Arg Cy ~ Pro Asn Hi ~ Glu Leu Gly Arg A ~ p Asn Glu Ply Gln Gln Ser Ala Pro Ala ser Hi ~ Leu Ile Arg Val Glu Gly AYn Asn Leu Ser Gln Tyr Val Aqp A ~ p Pro Thr Val Gly Arg Gln Ser Val Val Val Pro Tyr Glu Pro Pro Gln Val Gly Thr Glu Phe Thr Thr Leu Tyr Asn Phe Met Cy ~ Asn Ser Ser Cy ~ Val Gly Gly Met Asn Arg Arg Pro Ile Leu 260,265,270 Ile Ile Ile Thr Leu Glu Met Arg A ~ p Gly Gln Val Leu Gly Arg Arg Ser Phe Glu Gly Arg Cys Ala Cys Pro Gly Arg Arg Arg Ala Ala 2g0 295 300 CA 02245242 l998-07-28 WO 97/28186 PCTAFR97 / 0 ~ 214 Asp GlU Asp Hls Tyr Arg Glu Gln Gln Ala Leu Asn Glu Ser Ser Ala 305 ~ 10 315 320 Lys Asn Gly Ala Ala Ser Lys Arg Ala Phe Dy9 & ln Ser Pro Pro Ala Val Pro Ala Leu Gly Ala Gly Val Lys Lys Arg Arg Hl ~ Gly Asp Glu 340,345,350 Asp Thr Tyr Tyr Leu Gln Val Arg Gly Arg Glu Asn Phe Glu Ile Leu Met Lys Leu: Glu Ser Lys Leu Glu Leu Glu Leu Leuk Pro Gln Pro 370,375,380 Leu Val Asp Ser Tyr Arg Gln Gln Gln Gln Leu Leu Gln Arg Pro Ser His Leu Gln Pro Pro Ser Tyr Pro Gly Pro Leu Ser Pro Met Asn Lys Val His Gly Gly Met Asn Lys Leu Ser Val Asn Gln Leu Val Gly ~ i20 425 430 Gln ero Pro Pro ~ Ser Ser Ala Ala Thr Pro A ~ n Leu Gly Pro Val Gly Pro Gly Met Leu Asn Asn Hi ~ Gly Hi ~ Ala Pro Val Ala Asn Gly Glu ~ and Ser Ser Ser His Ser Ala Gln Ser Met Val Ser Gly Ser His Cys Thr Pro E'ro Pro Pro Tyr His Ala Asp Pro Ser Leu Val Arg Thr Trp Gly Pro (2) INFORMATION FOR SEQ ID NO: 20:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A ~ LENGTH: 17 pairs of base ~
(B) TYPE: nucleic acid ~ C) NUMBER OF BRINS: ~ imple (D) CONFIGURATION: linear (ii) TYPB OF MOLECULE: DNA
(iii) hY ~ OI ~ h ~ lQUE: NO
(iii) ANTI-SENSES: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 20:

(2) INFORMATION FOR SEQ ID NO: 21:

(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 19 pairs of ba ~ es (B) TYPE: Nucleic acid (C) NUMBER OF BRINS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(iii) ANTI-SENS: YES

WO 97/28186

7 ~ PCT ~ FR97 / 00214 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 21:

~ 2) INFORMATION FOR SEQ ID NO: 22:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 18 pairs of baAes (B) TYPE: nucleic acid (C) WOMBRE OF BRINS: ~ lmple (D) CONFIGURATION: linear tii) TYPE OF MOLECULE: DNA
(lii) ANTI-SENS: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: Z2:

(2) INFORMATION FOR SEQ ID NO: 23:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 18 pairY of ba eA
(B) TYPE: nucleic acid (C) NUMBER OF BRINS: 3imple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(iii) ANTI-SENS: YES

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 23:

(2) INFORMATION FOR SEQ ID NO: 24:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A ~ LENGTH: 21 pair ~ ba ~ e ~
(B TYPE: nucleic acid (C ~ NUMBER OF STRANDS: Yimple (CONFIGURATION: l ~ n ~ e (ii) TYPE OF MOLECULE: DNA ...
(iii) ANTI-SENSES: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 24:
GTCAACCAGC T ~ lG ~ GCCA G 2l (2) INFORMATION FOR SEQ ID NO: 25:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) NUMBER OF BRINS: ~ imple (D) CONFIGURATION: Lin ~ Aire (ii) TYPE OF MOLECULE: DNA
(iii) ANTI-SENS: YES

WO 97/28186 75 PCT ~ FR97 / OU214 (XI) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 25:
GTGGATCTCG GCCTCC l6 (2) INFORMATION FOR SEQ ID NO: 26:
(1) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 17 ~ pair of bases (B) TYPE: nucleic acid (C) NUMBER OF BRINS: ~ imple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(iii) ANTI-SENSES: NO

(XI) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 26:

(2) INFORMATION FOR SEQ ID NO: 27:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LON ~ U ~ UK: 19 pairs of baYe ~ -(B) TYPE: nucleic acid (C) NUMBER OF BRINS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(iii) ANTI-SENS: YES

(Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 27:

(2) INFORMATION FOR SEQ ID NO: 28:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A: LENGTH: 17 pairs of ba ~ e.
(B TYPE: nucleic acid (C ~ NUMBER OF 8RINS: qimP1e (D. CONE ~ IGURATION: lin ~ aire (ii) TYPE OF MOLECULE: DNA
(iii) ANTI-SENSES: NO

(XI) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 28:

(2) INFORMATION FOR SEQ ID NO: 29:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) L ~ N ~ u ~ uK: 18 pair ~ basic ~
(B) TYPE: nucleic acid rC) NOM8RE OF BRINS: simple (D) CONFIGURATION: lin ~ aire (ii) TYPE OF MOLECULE: DNA
(iii) ANTI-SENS: YES

- ~ Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 29:

(2) INFORMATION FOR SEQ ID NO: 30:
(1) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 18 ~ pair of bases (B) TYPE: nucleic acid (C) NUMBER OF BRINS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(iii) ANTI-SENSES: NO

(XI) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 30:

(2) INFORMATION FOR SEQ ID NO: 31:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LON ~ U ~ UK: 18 pair ~ of ba ~ e ~
(B) TYPE: nucleic acid (C) NUMBER OF STRANDS:
(D) CONFIGURATION: Linear (ii) TYPE OF MOLECULE: DNA
(1ii) ANTI-SENS: YES

(XI) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 31:

(2) INFORMATION FOR SEQ ID NO: 32:
(i) CHARACTERISTICS OF THE SEQUENCE:
A) LON ~ U ~ UK: 18 pairsY of ba ~ es ~ B) TYPE: nucleic acid ~ C) NUMBER OF BRINS: ~ imple ~ D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
~ 1ii) ANTI-SENS: NO

(XI) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 32:

(2) INFORMATION FOR SEQ ID NO: 33:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) L ~ N ~ U ~ UK: 19 pairs of ba ~ e ~
(B) TYPE: Nucleic acid (C) NUMBER OF BRINS: ~ imple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(iii) ANTI-SENS: YES

WO 97/28186 PCT ~ FR97 / 00214 - (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 33:
CcATCAGCTC CAGGcTcTc l9 (2) INFORMATION FOR SEQ ID NO: 39:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 18 pairs of basec (B) TYPE: nucleic acid (C) NUMBER OF BRINS: ~ lmple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(iii) ANTI-SEMS: YES

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 34:
CCAGG ~ CAGG CGCAGATG l8 (2) INFORMATION FOR SEQ ID NO: 35:
(i) CHARACTERISTICS OF THE SEQUENCE:
(AL LON'GUEUR: l9 pal ~ ba ~ and (8 'TYPE: nucleic acid.
(Cl NAME: BRE OF BRINS: simple (D, l CONFIGURATION: lin ~ area (ii) TYPE OF MOLECULE: DNA
(iii) ANTI-SENS: YES

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 35:
GATGAGGTGG CTCG ~ TG ~ A 19 (2) INFORMATION FOR SEQ ID NO: 36:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LO ~ U ~ UK: 19 pair ~ of ba ~ e ~
(B) TYPE: nucleic acid (C) NUMBER OF BRINS: ~ imple (D) CONFIGURATION: linear ~ ii) TYPE OF MOLECULE: DNA
(iii) ANTI-SENS: YES

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 36:
TGGTCAGGTT CTGCAGt; TG l9 (2) INFORMATION FOR SEQ ID NO: 37:
(i) CHARACTERXTICS OF THE SEQUENCE:
'A) LO ~ U ~ UK: 18 pairs of ba ~ ec B) TYP ~ S: Nucleic acid 'C) NAME OF BRINS: ~ imple , D) CONFIGURATION: li n ~ a ~ re (ii) TYPE OF MOLECULE: DNA
(iii) ANTI-SENSES: NO

WO 97/28186 PCTrFR97 / 00214 (X1) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 37:

(2) INFORMATION FOR SEQ ID NO: 38:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 21 pairs of ba3e3 (B) TYPE: aclde ~ ucleic (C) NUMBER OF BRINS: 3imple (D) CONFIGURATION: Linear (il) TYPE OF MOLECULE: DNA
(iil) ANTI-SENS: YES

(X1) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 38:
AGGAAAATAG AAGCGTCAGT C 2l (2) INFORMATION FOR SEQ ID NO: 39:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) L ~ N ~ u ~ u ~: 18 pair ~ of ba ~ e ~
(B) TYPE: nucleic acid (C) NUMBER OF BRINS: 3imple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(iii) ANTI-SENSES: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 39 :.
CAGGCCCACT TGCCTGCC l8 (2) INFORMATION FOR SEQ ID NO: 40:
(i) CHARACTERISTICS OF THE SEQUENCE:
(Al LENGTH: l9 pair3 of ba ~ e ~
(B TYPE: Nuclear acid (C NUMBER OF BRINS: imple (Dl CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(iii) ANTI-SENS: YES

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:
CTGTCCCCAA GCTGATGAG l9

Claims (36)

1. Purified polypeptide comprising an amino acid sequence chosen from:
a) the sequence SEQ ID No. 2;
b) the sequence SEQ ID No. 4;
c) the sequence SEQ ID No. 6;
d) the sequence SEQ ID No. 8;
e) the sequence SEQ ID No. 10;
f) the sequence SEQ ID No. 13;
g) the sequence SEQ ID No. 15;
h) the sequence SEQ ID No. 17;
i) the sequence SEQ ID No. 19;
and j) any biologically active sequence derived from SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, or SEQ ID No. 19. 2. Polypeptide according to claim 1, characterized in that it comprises the sequence of amino acids chosen from SEQ ID No. 6, SEQ ID No. 13, SEQ ID No. 15, SEQ ID
#17 and SEQ ID #19. 3. Polypeptide according to claim 1, characterized in that it comprises the sequence between:
- residue 110 and residue 310 of SEQ ID No. 2 or 6;
- residue 60 and residue 260 of SEQ ID No. 8. 4. Polypeptide according to claim 1, characterized in that it results from a splicing alternative of the messenger RNA of the corresponding gene. 5. Polypeptide according to any one of the preceding claims, characterized in that it is a recombinant polypeptide produced in the form of a protein of merger. 6. Isolated nucleic acid sequence coding for a polypeptide according to one any of the preceding claims. 7. Isolated nucleic acid sequence according to claim 6, characterized in that that it is chosen from:
a) the sequence SEQ ID No. 1;
b) the sequence SEQ ID No. 3;
c) the sequence SEQ ID No. 5;
d) the sequence SEQ ID No. 7;
e) the sequence SEQ ID No. 9;
f) the sequence SEQ ID No. 11;
g) the sequence SEQ ID No. 12;
h) the sequence SEQ ID No. 14;
i) the sequence SEQ ID No. 16;
j) the sequence SEQ ID No. 18;
k) nucleic acid sequences capable of hybridizing specifically to the sequence SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ
ID No. 11, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16 or SEQ ID No. 18 or their complementary sequences, or to hybridize specifically to their sequences proximal.;
and l) the sequences derived from the sequences a), b), c), d), e), f), g), h), i), j) or k) of fact of the degeneration of the genetic code, mutation, deletion, insertion, alternative splicing or allelic variability. 8. Nucleotide sequence according to claim 6, characterized in that it is of a sequence chosen from SEQ ID No. 5, SEQ ID No. 12, SEQ ID No. 14, SEQ ID
No. 16 and SEQ ID No. 18 respectively coding for the polypeptide of sequences SEQ ID No. 6, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17 and SEQ ID No. 19. 9. Cloning and/or expression vector containing an acid sequence nuclei according to any one of claims 6 to 8. 10. Vector according to claim 9, characterized in that it is the plasmid pSE1. 11. Host cell transfected with a vector according to claim 9 or 10. 12. Transfected host cell according to claim 11, characterized in that it is of E. coli MC 1061. 13. Nucleotide probe or nucleotide primer characterized in that it hybridizes specifically under stringent conditions with any of the sequences according to claims 6 to 8 or their complementary sequences or the corresponding messenger RNAs or the corresponding genes. 14. Probe or primer according to claim 13, characterized in that it comprises at least 16 nucleotides. 15. Probe or primer according to claim 13, characterized in that it comprises the entire sequence of the gene coding for one of the polypeptides of the claim 1. 16. Probe or nucleotide primer chosen from the following oligonucleotides or their complements:
SEQ ID No. 20: GCG AGC TGC CCT CGG AG
SEQ ID NO: 21: GGT TCT GCA GGT GAC TCA G
SEQ ID NO: 22: GCC ATG CCT GTC TAC AAG
SEQ ID No. 23: ACC AGC TGG TTG ACG GAG
SEQ ID No. 24: GTC AAC CAG CTG GTG GGC CAG
SEQ ID NO: 25: GTG GAT CTC GGC CTC C
SEQ ID No. 26: AGG CCG GCG TGG GGA AG
SEQ ID NO: 27: CTT GGC GAT CTG GCA GTA G
SEQ ID #28: GCG GCC ACG ACC GTG AC
SEQ ID NO: 29: GGC AGC TTG GGT CTC TGG
SEQ ID NO: 30: CTG TAC GTC GGT GAC CCC
SEQ ID NO: 31: TCA GTG GAT CTC GGC CTC
SEQ ID No. 32: AGG GGA CGC AGC GAA ACC
SEQ ID NO: 33: CCA TCA GCT CCA GGC TCT C
SEQ ID NO: 34: CCA GGA CAG GCG CAG ATG
SEQ ID NO: 35: GAT GAG GTG GCT GGC TGG A
SEQ ID NO: 36: TGG TCA GGT TCT GCA GGT G
SEQ ID No. 37: CAC CTA CTC CAG GGA TGC
SEQ ID No. 38: AGG AAA ATA GAA GCG TCA GTC
SEQ ID No. 39: CAG GCC CAC TTG CCT GCC
and SEQ ID NO: 40: CTG TCC CCA AGC TGA TGA G 17. Use of a sequence according to any one of claims 6 to 8 for manufacture of oligonucleotide primers for sequencing reactions or specific amplification according to the PCR technique or any variant of this one. 18. Couple of nucleotide primers, characterized in that it comprises the primers chosen from the following sequences:

a) forward primer: GCG AGC TGC CCT CGG AG (SEQ ID No. 20) antisense primer: GGT TCT GCA GGT GAC TCA G (SEQ ID No. 21) b) forward primer: GCC ATG CCT GTC TAC AAG (SEQ ID No. 22) antisense primer: ACC AGC TGG TTG ACG GAG (SEQ ID No. 23) c) forward primer: GTC AAC CAG CTG GTG GGC CAG (SEQ ID No. 24) reverse primer: GTG GAT CTC GGC CTC C (SEQ ID No. 25) d) forward primer: AGG CCG GCG TGG GGA AG (SEQ ID No. 26) reverse primer: CTT GGC GAT CTG.GCA GTA G (SEQ ID No. 27) e) forward primer: GCG GCC ACG ACC GTG A (SEQ ID No. 28) reverse primer: GGC AGC TTG GGT CTC TGG (SEQ ID No. 29) f) forward primer: CTG TAC GTC GGT GAC CCC (SEQ ID No. 30) reverse primer: TCA GTG GAT CTC GGC CTC (SEQ ID No. 31) g) forward primer: AGG GGA CGC AGC GM ACC (SEQ ID No. 32) reverse primer: GGC AGC TTG GGT CTC TGG (SEQ ID No. 29) h) direction primer: CCCCCCCCCCCCCCN (where N is equal to G, A or T) reverse primer: CCA TCA GCT CCA GGC TCT C (SEQ ID No. 33) i) direction primer: CCCCCCCCCCCCCCN (where N is equal to G, A or T) reverse primer: CCA GGA CAG GCG CAG ATG (SEQ ID No. 34) j) direction primer: CCCCCCCCCCCCCCCN (where N is equal to G, A or T) reverse primer: CTT GGC GAT CTG GCA GTA G (SEQ ID No. 27) k) sense primer: CAC CTA CTC CAG GGA TGC (SEQ ID No. 37) reverse primer: AGG AAA ATA GAA GCG TCA GTC (SEQ ID No. 38) and l) forward primer: CAG GCC CAC TTG CCT GCC (SEQ ID No. 39) antisense primer: CTG TCC CCA AGC TGA TGA G (SEQ ID No. 40). 19. Use of a sequence according to any one of claims 6 to 8, used in gene therapy. 20. Use of a sequence according to any one of claims 6 to 8, for the production of nucleotide probes or primers for diagnosis, or antisense sequences usable in gene therapy. 21. Use of nucleotide primers according to any one of claims 6 at 8 for sequencing. 22. Use of a probe or primer according to any one of claims 13 to 16, as an in vitro diagnostic tool for the detection, by experiments hybridization, nucleic acid sequences encoding a polypeptide according to any one of claims 1 to 4, in biological samples, or for highlighting aberrant syntheses or anomalies genetics. 23. In vitro diagnostic method for the detection of aberrant syntheses or genetic abnormalities in the nucleic acid sequences encoding for a polypeptide according to any one of claims 1 to 4, characterized in that it comprises:
- contacting a nucleotide probe according to any of the claims 13 to 16 with a biological sample under conditions allowing) the formation of a hybridization complex between said probe and the aforesaid nucleotide sequence, optionally after a preliminary step amplification of said nucleotide sequence, - the detection of the hybridization complex possibly formed;
- optionally the sequencing of the nucleotide sequence forming the hybridization complex with the probe of the invention. 24. Use of a nucleic acid sequence according to any of the claims 6 to 8, for the production of a recombinant polypeptide according to one any of claims 1 to 5. 25. Method for producing a recombinant protein SR-p70, characterized in that transfected cells according to claim 10 or 11 are cultured in conditions allowing the expression of a recombinant polypeptide of sequence SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, or SEQ ID No. 19 or any fragment or biologically active derivative, and that said recombinant polypeptide is recovered. 26. Mono or polyclonal antibodies or their fragments, chimeric antibodies or immunoconjugates, characterized in that they are capable of specifically recognizing a polypeptide according to any one of claims 1 to 4. 27. Use of the antibodies according to the preceding claim, for the purification or the detection of a polypeptide according to any one of claims 1 to 4 in a biological sample. 28. Process for the in vitro diagnosis of pathologies correlated to an expression or a abnormal accumulation of SR-p70 proteins, in particular the phenomena of cancerization, from a biological sample, characterized in that one brings into contact at least one antibody according to claim 25 with said biological sample, under conditions allowing the possible formation of specific immunological complexes between an SR-p70 protein and the or said antibodies and in that the specific immunological complexes are detected possibly formed. 29. Kit for the in vitro diagnosis of abnormal expression or accumulation of SR-p70 proteins in a biological sample and/or for the measurement of the rate expression thereof in said sample comprising:

- at least one antibody according to claim 25, optionally attached to a support, - means for revealing the formation of antigen/antibody complexes specific between an SR-p70 protein and said antibody and/or means of quantification of these complexes. 30. Method for early diagnosis of tumor formation characterized by what is shown in a sample of serum taken from a individual autoantibodies directed against an SR-p70 protein according to the steps consisting in bringing into contact a sample of serum taken from an individual with a polypeptide of the invention, optionally attached to a support, under conditions allowing the formation of specific immunological complexes between said polypeptide and the auto-antibodies possibly present in the serum sample, and in that the immunological complexes are detected specific eventually formed. 31. Method for determining an allelic variability, a mutation, a deletion, insertion, loss of heterozygosity or abnormality genetics of the SR-p70 gene characterized in that it uses at least one nucleotide sequence according to any one of claims 6 to 8. 32. Method for determining an allelic variability of the SR-p70 gene at the level of position -30 and -20 with respect to the initiation ATG of exon 2 which can be involved in pathologies and characterized in that it comprises at least:
- a step during which exon 2 is amplified by PCR
of the SR-p70 gene carrying the target sequence using primer pairs oligonucleotides according to any one of claims 6 to 8;
- a step during which the amplified products are treated with a restriction enzyme whose cleavage site corresponds to the allele sought;
- a step during which the detection or assay of at least one least one of the products of the enzymatic reaction. 33. Pharmaceutical composition comprising, as active principle, a polypeptide according to any one of claims 1 to 4. 34. Pharmaceutical composition according to the preceding claim, characterized in that it comprises a polypeptide according to claim 2. 35. Pharmaceutical composition containing an inhibitor or an activator of the activity of the SR-p70. 36. Pharmaceutical composition containing a polypeptide derived from a polypeptide according to any one of Claims 1 to 5, characterized in that it is a inhibitor or activator of SR-p70.