Patent Publication Number: US-6660848-B2

Title: Allelic variant of human STAT3

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     The present application is a divisional of application Ser. No. 09/526,542, now issued as U.S. Pat. No. 6,369,198 which is a continuation of International Application No. PCT/EP98/05844, filed Sep. 15, 1998, the entire contents of which are hereby incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to a human STAT 3  allelic variant, the cDNA sequence encoding it, its use in therapy and/or in diagnosis of autoimmune and/or inflammatory diseases, as well as pharmaceutical compositions comprising it. 
     BACKGROUND OF THE INVENTION 
     Signal Transducer and Activator of Transcription (STAT) proteins are a new class of intracellular transcription factors which play an essential function in the cellular responses to cytokines (Stahl et at., 1994; Gouilleux et at., 1995; Azam et al., 1995; Tian et at., 1994; May et al., 1996; and Iwatsuki et al., 1997). 
     Most of these proteins have been well characterized by sequencing, and their structure as well as the mechanism of their actions has been extensively analyzed and well documented (Wegenka et al., 1993; Akira et al., 1994; Wegenka et al., 1994; Quelle et al., 1995 and Silva et al., 1996). 
     These proteins contain SH 2  and SH 3  domains as well as a phosphorylation site at their carboxy-terminal region (Kapetein et al., 1996; and Herman et al., 1996). After cytokine receptor activation through ligand binding, the intracellular portion of the receptor becomes phosphorylated by an associated kinase of the JAK family. STAT proteins then bind to the phosphorylated receptor, through their SH 2  domain, and are in turn phosphorylated by JAKs (Stahl et al., 1995). Phosphorylated STAT proteins then dimerize and translocate to the nucleus, where they are able to recognize specific DNA responsive elements (Seidel et al., 1995; and Harroch et al., 1994). 
     STAT 3  has been identified as an important mediator of the signal imparted by the IL-6 family of cytokines, as well as by EGF and by a number of other interleukins and growth factors. 
     STAT 3  has been shown to play a central role in the upregulation of hepatic acute-phase proteins (Wegenka et al., 1993; and Zhang et al., 1996) in the growth arrest of monocytic cells (Yamanaka et al., 1996; and Minami et al., 1996) as well as in the survival of myeloma cells (Harroch et al., 1994). 
     DESCRIPTION OF THE INVENTION 
     During experiments on the analysis of STAT 3  interactions, we have amplified by RT-PCR from HepG2 cells a cDNA fragment corresponding to the SH 2  domain of human STAT 3 . We have found by DNA sequencing that the SH 2  domain we have isolated shows a divergence of 13 residues over the corresponding sequence of the original published human STAT 3  gene (Akira et al., 1994). 
     In order to determine the nature of this sequence variant, we have designed three pairs of primers with 3′ ends corresponding to nucleotide positions at variance between the two human cDNA sequences. 
     Upon such investigations it resulted that the new variant corresponds to at least the most frequent allele of human STAT 3 . 
     Therefore, the main object of this invention is the above-mentioned allelic variant of human STAT 3  protein. In particular, the object of the invention is a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a functionally equivalent salt, or a functionally equivalent derivative, or an active fraction, or a fusion protein. 
     The definition “salt” as used herein refers both to salts of the carboxyl-groups and to the salts of the amino functions of the compound obtainable through known methods. The salts of the carboxyl-groups comprise inorganic salts as, for example, sodium, potassium, calcium salts and salts with organic bases as those formed with an amine as triethanolamine, arginine or lisine. The salts of the amino groups comprise for example salts with inorganic acids as hydrochloric acid and with organic acids as acetic acid. 
     The definition “derivative” as herein used refers to derivatives which can be prepared from the functional groups present on the lateral chains of the amino acid moieties or on the terminal N- or C-groups according to known methods and are comprised in the invention when they are pharmaceutically acceptable i.e. when they do not destroy the protein activity or do not impart toxicity to the pharmaceutical compositions containing them. Such derivatives include for example esters or aliphatic amides of the carboxyl-groups and N-acyl derivatives of free amino groups or O-acyl derivatives of free hydroxyl-groups and are formed with acyl-groups as for example alcanoyl- or aroyl-groups. 
     As “active fraction” of the protein the present invention refers to any fragment or precursor of the polypeptidic chain of the compound itself, alone or in combination with related molecules or residues bound to it, for example residues of sugars or phosphates, or aggregates of the polypeptide molecule when such fragments or precursors show the same activity of the protein of the invention, as medicament. 
     The definition “fusion protein” as herein used refers to polypeptides comprising the polypeptide of the invention above specified fused with another protein and having a longer lasting half-life in body fluids. It can for example be fused with another protein such as, for example, an immunoglobulin. 
     Another object of the invention is the DNA molecule comprising the DNA sequence coding for the allelic variant of the invention, including nucleotide sequences substantially the same. 
     “Nucleotide sequences substantially the same” includes all other nucleic acid sequences which, by virtue of the degeneracy of the genetic code, also code for the given amino acid sequences. In particular, the present invention refers to the nucleotide sequence comprising the SEQ ID NO:1. 
     The present invention also refers to recombinant DNA molecules which hybridize with the DNA sequence coding for the above-mentioned allelic variant of hSTAT 3  and whose nucleotide sequences show at least the same 13 differences in the SH 2  domain (with respect to the human STAT 3  sequence in Akira et al., 1994), as shown in FIG.  1 . The gene can contain, or not, the natural introns and can be obtained for example by extraction from appropriate cells and purification with known methods. 
     Furthermore, the present invention also includes recombinant DNA molecules which hybridize under stringent conditions with a probe having a nucleotide sequence selected between SEQ ID NO:16 and SEQ ID NO:17. 
     The term “stringent conditions” refers to hybridization and subsequent washing conditions which those of ordinary skill in the art conventionally refer to as “stringent”. See Ausubel et al.,  Current Protocols in Molecular Biologic supra. Interscience . N.Y. pare. 6.3 and 6.4 (1987, 1992), and Sambrook et al, 1989. Without limitation, examples of stringent conditions include washing conditions 12-20° C. below the calculated Tm of the hybrid under study in, e.g. 2×SSC and 0.5% SDS for 5 minutes, 2×SSC and 0.1% SDS for 15 minutes; 0.1×SSC and 0.5% SDS at 37° C. for 30-60 minutes and then a 0.1×SSC and 0.5% SDS at 68° C. for 30-60 minutes. Those of ordinary skill in this art understand that stringency conditions also depend on the length of the DNA sequences, oligonucleotide probes (such as 10-40 bases) or mixed oligonucleotide probes. If mixed probes are used, it is preferable to use tetramethyl ammonium chloride (TMAC) instead of SSC. See  Ausubel . supra. 
     The invention also includes expression vectors which comprise the above DNAs, host-cells transformed with such vectors and a process of preparation of such allelic variant of hSTAT 3 , its active fragments or fusion proteins, through the culture in appropriate culture media of said transformed cells. 
     The DNA sequence coding for the protein of the invention can be inserted and ligated into a suitable plasmid. Once formed, the expression vector is introduced into a suitable host cell, which then expresses the vector(s) to yield the desired protein. 
     Expression of any of the recombinant proteins of the invention as mentioned herein can be effected in eukaryotic cells (e.g. yeasts, insect or mammalian cells) or prokaryotic cells, using the appropriate expression vectors. Any method known in the art can be employed. 
     For example the DNA molecules coding for the proteins obtained by any of the above methods are inserted into appropriately constructed expression vectors by techniques well known in the art (see Sambrook et al, 1989). Double stranded cDNA is linked to plasmid vectors by homopolymeric tailing or by restriction linking involving the use of synthetic DNA linkers or blunt-ended ligation techniques: DNA ligases are used to ligate the DNA molecules and undesirable joining is avoided by treatment with alkaline phosphatase. 
     In order to be capable of expressing the desired protein, an expression vector should comprise also specific nucleotide sequences containing transcriptional and translational regulatory information linked to the DNA coding the desired protein in such a way as to permit gene expression and production of the protein. First in order for the gene to be transcribed, it must be preceded by a promoter recognizable by RNA polymerase, to which the polymerase binds and thus initiates the transcription process. There are a variety of such promoters in use, which work with different efficiencies (strong and weak promoters). 
     For eukaryotic hosts, different transcriptional and translational regulatory sequences may be employed, depending on the nature of the host. They may be derived form viral sources, such as adenovirus, bovine papilloma virus, Simian virus or the like, where the regulatory signals are associated with a particular gene which has a high level of expression. Examples are the TK promoter of the Herpes virus, the SV 40  early promoter, the yeast gal 4  gene promoter, etc. Transcriptional initiation regulatory signals may be selected which allow for repression and activation, so that expression of the genes can be modulated. 
     The DNA molecule comprising the nucleotide sequence coding for the protein of the invention is inserted into vector(s), having the operably linked transcriptional and translational regulatory signals, which is capable of integrating the desired gene sequences into the host cell. 
     The cells which have been stably transformed by the introduced DNA can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may also provide for phototrophy to a auxotropic host, biocide resistance, e.g. antibiotics, or heavy metals such as copper, or the like. The selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of proteins of the invention. 
     Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells, that contain the vector may be recognized and selected form those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to “shuttle” the vector between host cells of different species. 
     Once the vector(s) or DNA sequence containing the construct(s) has been prepared for expression the DNA construct(s) mat be introduced into an appropriate host cell by any of a variety of suitable means: transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate-precipitation, direct microinjection, etc. 
     Host cells may be either prokaryotic or eukaryotic. Preferred are eukaryotic hosts, e.g. mammalian cells, such as human, monkey, mouse, and Chinese hamster ovary (CHO) cells, because they provide post-translational modifications to protein molecules, including correct folding or glycosylation at correct sites. Also yeast cells can carry out post-translational peptide modifications including glycosylation. A number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number of plasmids which can be utilized for production of the desired proteins in yeast. Yeast recognizes leader sequences on cloned mammalian gene products and secretes peptides bearing leader sequences (i.e., pre-peptides). 
     After the introduction of the vector(s), the host cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of the desired proteins. 
     Purification of the recombinant proteins is carried out by any one of the methods known for this purpose, i.e. any conventional procedure involving extraction, precipitation, chromatography, electrophoresis, or the like. A further purification procedure that may be used in preference for purifying the protein of the invention is affinity chromatography using monoclonal antibodies which bind the target protein and which are produced and immobilized on a gel matrix contained within a column. Impure preparations containing the recombinant protein are passed through the column. The protein will be bound to the column by the specific antibody while the impurities will pass through. After washing, the protein is eluted from the gel by a change in pH or ionic strength. 
     As already stated, the protein of the invention is useful in the therapy and/or diagnosis of autoimmune and/or inflammatory diseases. Therefore, in a further aspect, the present invention provides the use of the protein of the invention in the manufacture of a medicament for the treatment of autoimmune diseases and/or inflammatory diseases. 
     The medicament is preferably presented in the form of a pharmaceutical composition comprising the protein of the invention together with one or more pharmaceutically acceptable carriers and/or excipients. Such pharmaceutical compositions form yet a further aspect of the present invention. 
     The invention will now be described by means of the following Examples, which should not be construed as in any way limiting the present invention. The Examples will refer to the Figures specified here below. 
    
    
     DESCRIPTION OF THE FIGURES 
     FIG. 1 shows a comparison of the EMBL-GB-deposited cDNA sequence of the SH 2  domain of human STAT (SEQ ID NO:3) with the corresponding human HepG2 (nucleotides 1689-2112 of SEQ ID NO:1) and mouse liver (SEQ ID NO:5) CDNA fragments. The shown 424 bp nucleotide sequence and its numbering are from the SH 2  domain of the human STAT 3  CDNA sequence deposited in the EMBL-GB databases (Akira et al., 1994). Nucleotides at variance identified in human HepG2 (this patent application) and mouse liver cDNAs (Akira et al., 1994) are reported above the full sequence, in bold and underlined. Also in bold and underlined on the full sequence is indicated the nucleotide change resulting in a variation at the amino acid level, a Leucine coded in the deposited sequence being substituted by a Valine in the new sequence of this patent application. Amino acid sequences encoded by the hEMDL and m liver cDNAs are SEQ ID NOs:4 and 6, respectively. Primers US 0 ; LS 0 ; US 1 ; LS 1 ; LS 2 ; US 3 ; LS 3 ; US 4  and LS 4 , used in RT-PCR reactions are indicated by bold arrowhead above the sequences. 
     FIG. 2A shows the sequencing strategy and FIGS. 2B and 2C report the complete nucleotide sequence of human STAT 3  (SEQ ID NO:1) isolated from human HepG2 cells, in particular the coding region. The nucleotides at variance with the known published sequence are shown in bold and underlined. Amino acid residues modified with respect to the published sequence are shown below the nucleotide sequence. 
     FIGS. 3A-3D show the analysis of the expression of the originally published hSTAT 3  and the new variant hSTAT 3  cDNAs. RNA was extracted with the Trizol regent, reverse-transcribed with oligo. (dT) and the analytical PCR reaction was carried out with the Tag polymerase in capillary tubes, as described in the Examples. FIGS. 3A and 3C show PCR products amplified with the US 1 /LS 1  pair of primers specific for the original published hSTAT 3  sequence, and FIGS. 3B and 3D show PCR products amplified with the US 3 /LS 3  pair of primers specific for the new hSTAT 3  sequence variant found in this patent application. The lanes are as follows: M) Molecular size markers. 1) Liver RNA. 2) Spleen RNA. 3) Uterus RNA. 4) Lung RNA. 5) Skin RNA. 6) RNA from cord blood cells. 7) Dermal fibroblasts RNA. 8) Heart RNA with no reverse transcriptase. 9) Heart RNA. 10) Fetal liver RNA with no reverse transcriptase. 11) Fetal liver RNA. 12) Small intestine RNA with no reverse transcriptase. 13) Small intestine RNA. 14) Placental RNA with no reverse transcriptase. 15) Placental RNA. 
     FIGS. 4A and 4B show the amplification of an artificial DNA template with primers US 1 /LS 1 . The artificial DNA template composed of the hSTAT 3  variant sequence fragment flanked at its 5′ end by the US 1  primer sequence and at its 3′ end by the LS 1  primer sequence, was created by preparative PCR, using primers US 4 /LS 4 , from HepG2 cDNA (where only the variant sequence could be amplified, not shown), as described in the Materials and methods section. The artificial template was fractionated in 2% agarose gel and the relevant band of 285 bp was purified with the agarose gel DNA extraction kit (Boeringer Mannheim, Mannheim, Germany). This template was then spiked at various concentrations to 1 μl of the relevant cDNA (originated from approximately 100 ng of the corresponding RNA). Lanes: M) Molecular size markers. 1) No spiking. 2) 0.3 fg of artificial template spiked. 3) 3 fg of artificial template spiked. 4) 30 fg of artificial template spiked. 5) 300 fg of artificial template spiked. 
     FIG. 5 shows the PCR analysis of the original hSTAT 3  and the new variant hSTAT 3  genomic sequence fragment. 40 ng of human genomic DNA were used in analytical PCR reactions carried out in capillary tubes, as described in the Materials and methods section. Lanes: M) Molecular size markers. 2, 4) Genomic DNA amplified with the US 1 /LS 1  pair of primers, specific for the original, published hSTAT 3  sequence. 2) Genomic DNA amplified with the US 1 /LS 2  pair of primers, specific for the original, published hSTAT 3  sequence. 3) Genomic DNA amplified with the US 3 /LS 3  pair of primers specific for the new variant hSTAT 3  sequence. 5, 6, 7, 8) Genomic DNA amplified with the US 1 /LS 1  pair of primers and spiked with 0.3, 3, 30 and 300 fg respectively, of the US 4 /LS 4 -amplified artificial DNA template. 
    
    
     EXAMPLES 
     MATERIALS AND METHODS 
     Materials 
     HepG2 human hepatoma cell line was from ATCC (Rockville, Md., USA). Total human RNA from heart, liver, fetal liver, small intestine placenta and human genomic DNA were obtained from Clontech (Palo Alto, Calif., USA). Other RNAs used in this patent application were prepared in our laboratory. 
     Pfu polymerase was from Stratagene (La Jolla, Calif., USA); DNA Tag polymerase was from Advanced Biotechnology, Leatherhead, UK. DNA Sequencing Kit was from Perkin Elmer (Applied Biosystems Division, Foster City, Calif., USA); SuperScript II reverse transcriptase (200 U/μl) and Trizol Reagent for RNA extraction were from Gibco (Grand Island, N.Y., USA). 
     Oligonucleotide primers 
     All primers used in this patent application were designed in our laboratory using the software OLIGO (National Biosciences, Plymouth, Minn., USA), in order to optimize the specificity of PCR amplification of template nucleotide sequences differing by only one or few nucleotides. 
     All primers were synthesized in our laboratory, with a 392 DNA/RNA Synthesizer from Perkin Elmer (Applied Biosystems Division, Foster City, Calif., USA). A first pair of primers, US 0 /LS 0 , was used for the isolation of 424 bp containing the whole SH 2  domain of human STAT 3  cDNA. 
     The nucleotide sequences of all the primers used are shown below. 
     Primer USO 5′ AAC ACC ATG GCC TGG CTA GAC AAT ATC ATC GAC CT SEQ ID NO: 7 GTG AAA AAG TA 3′ 
     Primer LSO 5′ ATA TAT GGA TCC TGG GGC AGC GCT ACC TGG GTC AGC SEQ ID NO: 8 TTC 3′ 
     Primer STAU 5′ TCC CCG GAA GCT TCA CAC GCG CAG CCC CGG CTT CT 3′ SEQ ID NO: 9 
     Primer STAL 5′ GTT CAT CAC TTT TGT GTT TGT GCC CAG AAT 3′ SEQ ID NO: 10 
     Primer STBU 5′ GAC AAA GAC TCT GGG GAC GTT GCA GCT CTC 3′ SEQ ID NO: 11 
     Primer STBL 5′ TCA GTC CTC GAG TAT CTT TCT GCA GCT TCC GTT CT 3′ SEQ ID NO: 12 
     Primer US 1  5′ TGA AGG GTA CAT CAT GGG TTT C 3′ SEQ ID NO: 13 
     Primer LS 1  5′ TCA GGA TAG AGA TAG ACA AGT GGA GAC AA 3′ SEQ ID NO: 14 
     Primer LS 2  5° CCT CCT TCT TTG CTG CTT TCA CTG AAG 3′ SEQ ID NO: 15 
     Primer US 3  5° CGA AGG GTA CAT CAT GGG CTT T 3′ SEQ ID NO: 16 
     Primer LS 3  5° CCT CCT TCT TTG CTG CTT TCA CTG AAT CTT 3′ SEQ ID NO: 17 
     Primer US 4  5′ TGA AGG GTA CAT CAT GGG TTT CAT CAG TAA GGA 3′ SEQ ID NO: 18 
     Primer LS 4  5′ TCA GGA TAG AGA TAG ACA AGT GGA GAC AAC AGG ATA T 3′ SEQ ID NO: 19 
     The position of primers USO/LSO in the hSTAT 3  sequence is shown in FIG.  1 . 
     Additional primers for isolating the entire human STAT 3  cDNA were: Primer STAU, Primer STAL, Primer STBU and Primer STBL. 
     Two additional primer pairs, called US 1 /LS 1  and US 1 /LS 2 , amplifying products of 285 and 111 bp respectively, were uniquely specific for the original published sequence of human STAT 3  cDNA (Akira et al., 1994), but not for the hSTAT 3  variant sequence we have found in this patent application. At least one nucleotide at variance between the published and the variant STAT 3  sequences was positioned at the 3′ end of each primer. 
     The US 3 /LS 3  pair of primers was uniquely specific for the variant hSTAT 3  sequence described in this patent application. This US 3 /LS 3  pair of primers did amplify a 111 bp fragment specifically in the hSTAT 3  variant sequence, corresponding to the sequence amplified by the US 1 /LS 2  primers in the original published hSTAT 3  cDNA sequence. 
     A validation pair of primers, US 4 /LS 4 , to create an artificial hSTAT 3  template of 285 bp corresponding to the expected product of primers US 1 /LS 1 , has been used. 
     RNA and RT-PCR 
     Total RNA from human HepG2 cells was prepared by the method of Bimboim (Bimboim, 1988). For other tissues and cells, RNA was extracted with the Trizol reagent available from Gibco, Grand Island, N.Y., USA, following manufacturer instructions. 
     Oligo(dT) was used to prime reverse transcription of 5 μg of total RNA with 200 U of SuperScript II reverse transcriptase (RT) in 50 μl reaction mixture. The RT reaction was carried out at 37° C. for 1h and 30 min. Preparative PCR was then performed using the RT products as the cDNA templates. PCR reactions contained 10 μl of cDNA, 50 pmoles of each primer (see below), 2.5 units of Stratagene Pfu polymerase, 0.2 mM of each of the four deoxynucleotide triphosphates, 10 μl of Pfu buffer, in a reaction volume of 100 μl, overlaid with 50 μl of mineral oil. 
     Amplification was usually performed for 30 cycles with a temperature profile of 45 seconds at 94° C. (denaturation), 45 seconds at 50 to 60° C. (annealing) and 5 minutes at 72° C. (exention). PCR products were purified by centrifugation through Microcon 100 filters (Amicon) and then subjected to electrophoresis on 1.5% agarose gels in Tris/borate/EDTA buffer. Analytical PCRs were performed in capillary tubes, with the same concentration of reagents described above in ten-fold less volume, except for the Pfu polymerase which was substituted with 0.5 U of Tag polymerase. The temperature profile was 94° C. for denaturation, 55° C. for annealing and 72° C. for extention. 
     DNA sequencing 
     STAT 3  PCR products were sequenced as depicted in FIG.  2 . DNA sequences were performed with the dideoxy method, using a DNA sequencing kit (Perkin Elmer, Applied Biosystems Division, Foster City, Calif., USA) on an ABI model 373A automated sequencer, following manufacturer instructions. The nucleotide sequences of all cDNA fragments were determined from sequencing both DNA strands. Nucleotide and deduced amino acid sequences were compared with those in GenBank and the Swiss-Prot database. 
     Results and Discussion Isolation and Sequencing of a cDNA Fragment Coding for Human STAT 3   
     In order to isolate the SH 2  domain of human STAT 3 , we have amplified by RT-PCR, a cDNA fragment of 424 base pairs corresponding to nucleotide positions between 1909 and 2332 of the published human placental STAT 3  cDNA sequence (Akira et al., 1994), using total RNA from HepG2 cells. 
     This PCR fragment was then inserted in an expression vector, and the nucleotide sequence was determined. Results (FIG. 1) showed that 13 nucleotide residues differed from the original human placental cDNA sequence. The majority of the 13 modified residues were located in third codon position, resulting in no change of the corresponding amino acid sequence. 
     Only one mutated nucleotide residue resulted in the substitution of a leucine at position  667  in the human STAT 3  protein with a valine (FIG.  1 ). We have then amplified from HepG2 cells two additional cDNA fragments corresponding to the whole coding region of the human STAT 3  cDNA. Sequencing of these fragments (FIG. 2) showed that overall 43 nucleotide were at variance with the published sequence, corresponding to a total of 6 amino acid changes (Akira et al., 1994). 
     The published human and mouse STAT 3  consensus sequences are known to differ by 172 nucleotides, while the new human STAT 3  sequence we present here differs by 193 residues with the mouse sequence (Raz et al., 1994; Akira et al., 1994; Zhong et al., 1994). 
     Thus, at the nucleotide level, the new human STAT 3  sequence results in a slightly increased evolutionary distance with the mouse sequence. A region ranging between nucleotides 1680 and 1940 of the original human sequence showed a high nucleotide conservation between man and mouse. Such conservation is lost when the new human sequence presented in this patent application is considered. 
     On the contrary, at the amino acid level the new human sequence is more closely related to the mouse sequence. All six changes in the new human STAT 3  amino acid sequence return the corresponding original mouse (and rat) residues, so that only one residue is now at variance between the human and the 770 amino acids consensus sequence of mouse STAT 3 : a glutamic acid at position  760  of the human sequence is substitued with an aspartic acid in the mouse sequence. The encoded STAT 3  sequence therefore now results as one of the most conserved among known genetic determinants. As a reference, mouse and human STAT 1  and STAT 5  proteins differ by 67 and 29 amino acid residues, respectively. 
     STAT 3 , like other STAT family members, is known to bind several different proteins in order to accomplish its multiple functions (Damell, 1997). The SH 2  domain of STAT 3  interacts with the intracellular portion of signal transducing receptor molecules, while the C-terminal region is important for activation and dimerization (Sasse et al., 1997), and the central region is important for DNA binding (Horvath et al., 1995). 
     Among the six amino acid changes described in the present patent application, one falls within the N-terminal region, at position  288 . The second amino acid change falls at position  460 , in the DNA-binding domain. Two additional changes fall within the SH 3  domain, at position  548  and  561  respectively. 
     Finally, two more amino acid changes fall within the SH 2  domain at position  667  and in the C-terminal region, at position  730  respectively (see FIG.  2 ). 
     Characterization of the New STAT 3  Sequence Variant 
     In order to determine the nature of the new sequence variant presented here, we have designed three pairs of primers with 3′ ends corresponding to nucleotide positions at variance between the two human cDNA sequences. The first and the second pair of primers (US 1 /LS 1  and US 1 /LS 2 ) were exclusively specific for the original published nucleotide sequence of the hSTAT 3  cDNA, while the third pair of primers (US 3 /LS 3 ) was exclusively specific for the new variant human STAT 3  nucleotide sequence we have determined. 
     We have used the two primer pairs US 1 /LS 1  and US 3 /LS 3  (specific for the original and the new variant sequences respectively) to amplify RNAs from 11 different human tissues in 22 separate RT-PCR reactions. Each RNA source we have examined was derived from pools of 1 to 17 individuals, with a total of 31 individuals analyzed. 
     Since the original hSTAT 3  cDNA sequence was derived from human placenta, this tissue was included among the 11 RNA sources tested. As shown in FIG. 3, only the pair of primers specific for the new sequence variant were able to amplify all the eleven RNAs tested, resulting in the expected amplification product, while no significant band could be obtained in any RNA tested with the primers corresponding to the original published hSTAT 3  sequence. Since the US 1 /LS 1  primers did not result in any significant amplification product, we wanted to verify whether this failure was due to a defect in the primers, either in their intrinsic ability to anneal to the appropriate template, or in their ability to prime the amplification reaction. 
     In other words, we wanted to validate the US 1 /LS 1  pair of primers. Validation primers US 4 /LS 4  were thus designed to match exactly primers US 1 /LS 1 , but each primer with a 3′ extention matching the hSTAT 3  variant sequence determined in this work. 
     Amplification would then result in an artificial hybrid template composed of the hSTAT 3  variant sequence fragment, with its 5′ and 3′ ends identical to primers US 1  and LS 1  respectively. This artificial template should allow effective amplification with primers US 1 /LS 1 , even in the absence of the corresponding natural DNA template (i.e., the original, published hSTAT 3  cDNA fragment of 285 bp). 
     This artificial template was obtained by PCR with primers US 4 /LS 4 , and spiked at different concentrations in human placental cDNA and in other human cDNAs. Primers US 1 /LS 1  were then used to amplify these spiked cDNAs, and a PCR product of the expected size was readly obtained (FIG.  4 ); This result therefore excluded a failure of the US 1 /LS 1  pair of primers in the amplification reaction. 
     We have then used the US 1 /LS 1 , US 1 /LS 2  and US 3 /LS 3  pairs of primers to amplify human genomic DNA. The expected amplification product was again obtained only with the primer pair specific for the new variant sequence we have determined (FIG.  5 ). 
     We have shown that the mouse and the revised human STAT 3  protein sequences are highly conserved, with only one residue being at variance between the two species over 770 amino acid residues of total length. 
     We could not detect the hSTAT 3  nucleotide sequence originally described by Akira et al. (Akira et al., 1994) in any of the human genomic or cDNA sources we have tested. The original published nucleotide sequence and the new sequence variant are not therefore different genes or splice variants contemporaneously present in the same genome, since only one sequence (the one identified in this patent application) was detected in each human nucleic acid source tested. The two hSTAT 3  sequence variants could be different alleles. 
     In this case however, the new variant sequence is likely to be predominant, since it was represented in all nucleic acid samples tested, derived from a total of 31 individuals. The original published hSTAT 3  sequence was not represented at all in these individuals. 
     References 
     1. Akira, S., et al., (1994) Cell 77, 63-71; 
     2. Azam, M., et al., (1995) EMBO Journal 14, 1402-1411; 
     3. Bimboim, H. C. (1988) Nucleic Acids Research 16, 1487-1497; 
     4. Darnell J. E. (1997) Science 277, 1630-1635; 
     5. Gouilleux, F., et al., (1995) EMBO Journal 14, 2005-2013; 
     6. Gram, H., et al., (1992) Proceedings of the National Academy of Sciences of the United States of America 89, 3576-3580; 
     7. Harroch, S., et al., (1994) Journal of Biological Chemistry 269, 26191-26195; 
     8. Hemmann, U., et al., (1996) Journal of Biological Chemistry 271, 12999-13007; 
     9. Horvath C. M. et al., (1995) Genes &amp; Development 9, 984-994; 
     10. Iwatsuki, K., et al., (1997) J. Biol. Chem. 272, 8149-8152; 
     11. Kapetein, A., et al., (1996) Journal of Biological Chemistry 271, 5961-5964; 
     12. May, P., et al., (1996) FEBS Lett. 394, 221-226; 
     13. Minami, M., et al., (1996) Proceedings of the National Academy of Sciences of the United States of America 93, 3963-3966; 
     14. Quelle, F. W., et al., (1995) Molecular &amp; Cellular Biology 15, 3336-3343; 
     15. Raz, R., et al., (1994) Journal of Biological Chemistry 269, 24391-24395; 
     16. Sasse J. et al., (1997) Mol. Cell Biol. 17, 46774686; 
     17. Seidel, H. M., et al., (1995) Proceedings of the National Academy of Sciences of the United States of America 92, 3041-3045; 
     18. Shi, W., et al., (1996) International Immunology 8, 1205-1211; 
     19. Silva, C. M., et al., (1996) Molecular Endocrinology 10, 508-518; 
     20. Stahl, N., et al., (1994) Science 263, 92-95; 
     21. Stahl, N., et al., (1995) Science 267, 1349-1353; 
     22. Tian, S. S., et al., (1994) Blood 84, 1760-1764; 
     23. Wegenka, U. M., et al., (1993) Mol. Cell Biol. 13, 276-288; 
     24. Wegenka, U. M., et al., (1994) Molecular &amp; Cellular Biology 14, 3186-3196; 
     25. Yamanaka, Y., et al., (1996) EMBO Journal 15, 1557-1565; 
     26. Zhang, D., et al., (1996) Journal of Biological Chemistry 271, 9503-9509; 
     27. Zhong, Z., et al., (1994) Science 264, 95-98. 
     
       
         
           
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            1
atg gcc caa tgg aat cag cta cag cag ctt gac aca cgg tac ctg gag       48
Met Ala Gln Trp Asn Gln Leu Gln Gln Leu Asp Thr Arg Tyr Leu Glu
1               5                   10                  15
cag ctc cat cag ctc tac agt gac agc ttc cca atg gag ctg cgg cag       96
Gln Leu His Gln Leu Tyr Ser Asp Ser Phe Pro Met Glu Leu Arg Gln
            20                  25                  30
ttt ctg gcc cct tgg att gag agt caa gat tgg gca tat gcg gcc agc      144
Phe Leu Ala Pro Trp Ile Glu Ser Gln Asp Trp Ala Tyr Ala Ala Ser
        35                  40                  45
aaa gaa tca cat gcc act ttg gtg ttt cat aat ctc ctg gga gag att      192
Lys Glu Ser His Ala Thr Leu Val Phe His Asn Leu Leu Gly Glu Ile
    50                  55                  60
gac cag cag tat agc cgc ttc ctg caa gag tcg aat gtt ctc tat cag      240
Asp Gln Gln Tyr Ser Arg Phe Leu Gln Glu Ser Asn Val Leu Tyr Gln
65                  70                  75                  80
cac aat cta cga aga atc aag cag ttt ctt cag agc agg tat ctt gag      288
His Asn Leu Arg Arg Ile Lys Gln Phe Leu Gln Ser Arg Tyr Leu Glu
                85                  90                  95
aag cca atg gag att gcc cgg att gtg gcc cgg tgc ctg tgg gaa gaa      336
Lys Pro Met Glu Ile Ala Arg Ile Val Ala Arg Cys Leu Trp Glu Glu
            100                 105                 110
tca cgc ctt cta cag act gca gcc act gcg gcc cag caa ggg ggc cag      384
Ser Arg Leu Leu Gln Thr Ala Ala Thr Ala Ala Gln Gln Gly Gly Gln
        115                 120                 125
gcc aac cac ccc aca gca gcc gtg gtg acg gag aag cag cag atg ctg      432
Ala Asn His Pro Thr Ala Ala Val Val Thr Glu Lys Gln Gln Met Leu
    130                 135                 140
gag cag cac ctt cag gat gtc cgg aag aga gtg cag gat cta gaa cag      480
Glu Gln His Leu Gln Asp Val Arg Lys Arg Val Gln Asp Leu Glu Gln
145                 150                 155                 160
aaa atg aaa gtg gta gag aat ctc cag gat gac ttt gat ttc aac tat      528
Lys Met Lys Val Val Glu Asn Leu Gln Asp Asp Phe Asp Phe Asn Tyr
                165                 170                 175
aaa acc ctc aag agt caa gga gac atg caa gat ctg aat gga aac aac      576
Lys Thr Leu Lys Ser Gln Gly Asp Met Gln Asp Leu Asn Gly Asn Asn
            180                 185                 190
cag tca gtg acc agg cag aag atg cag cag ctg gaa cag atg ctc act      624
Gln Ser Val Thr Arg Gln Lys Met Gln Gln Leu Glu Gln Met Leu Thr
        195                 200                 205
gcg ctg gac cag atg cgg aga agc atc gtg agt gag ctg gcg ggg ctt      672
Ala Leu Asp Gln Met Arg Arg Ser Ile Val Ser Glu Leu Ala Gly Leu
    210                 215                 220
ttg tca gcg atg gag tac gtg cag aaa act ctc acg gac gag gag ctg      720
Leu Ser Ala Met Glu Tyr Val Gln Lys Thr Leu Thr Asp Glu Glu Leu
225                 230                 235                 240
gct gac tgg aag agg cgg caa cag att gcc tgc att gga ggc ccg ccc      768
Ala Asp Trp Lys Arg Arg Gln Gln Ile Ala Cys Ile Gly Gly Pro Pro
                245                 250                 255
aac atc tgc cta gat cgg cta gaa aac tgg ata acg tca tta gca gaa      816
Asn Ile Cys Leu Asp Arg Leu Glu Asn Trp Ile Thr Ser Leu Ala Glu
            260                 265                 270
tct caa ctt cag acc cgt caa caa att aag aaa ctg gag gag ttg cag      864
Ser Gln Leu Gln Thr Arg Gln Gln Ile Lys Lys Leu Glu Glu Leu Gln
        275                 280                 285
caa aaa gtt tcc tac aaa ggg gac ccc att gta cag cac cgg ccg atg      912
Gln Lys Val Ser Tyr Lys Gly Asp Pro Ile Val Gln His Arg Pro Met
    290                 295                 300
ctg gag gag aga atc gtg gag ctg ttt aga aac tta atg aaa agt gcc      960
Leu Glu Glu Arg Ile Val Glu Leu Phe Arg Asn Leu Met Lys Ser Ala
305                 310                 315                 320
ttt gtg gtg gag cgg cag ccc tgc atg ccc atg cat cct gac cgg ccc     1008
Phe Val Val Glu Arg Gln Pro Cys Met Pro Met His Pro Asp Arg Pro
                325                 330                 335
ctc gtc atc aag acc ggc gtc cag ttc act act aaa gtc agg ttg ctg     1056
Leu Val Ile Lys Thr Gly Val Gln Phe Thr Thr Lys Val Arg Leu Leu
            340                 345                 350
gtc aaa ttc cct gag ttg aat tat cag ctt aaa att aaa gtg tgc att     1104
Val Lys Phe Pro Glu Leu Asn Tyr Gln Leu Lys Ile Lys Val Cys Ile
        355                 360                 365
gac aaa gac tct ggg gac gtt gca gct ctc aga gga tcc cgg aaa ttt     1152
Asp Lys Asp Ser Gly Asp Val Ala Ala Leu Arg Gly Ser Arg Lys Phe
    370                 375                 380
aac att ctg ggc aca aac aca aaa gtg atg aac atg gaa gaa tcc aac     1200
Asn Ile Leu Gly Thr Asn Thr Lys Val Met Asn Met Glu Glu Ser Asn
385                 390                 395                 400
aac ggc agc ctc tct gca gaa ttc aaa cac ttg acc ctg agg gag cag     1248
Asn Gly Ser Leu Ser Ala Glu Phe Lys His Leu Thr Leu Arg Glu Gln
                405                 410                 415
aga tgt ggg aat ggg ggc cga gcc aat tgt gat gct tcc ctg att gtg     1296
Arg Cys Gly Asn Gly Gly Arg Ala Asn Cys Asp Ala Ser Leu Ile Val
            420                 425                 430
act gag gag ctg cac ctg atc acc ttt gag acc gag gtg tat cac caa     1344
Thr Glu Glu Leu His Leu Ile Thr Phe Glu Thr Glu Val Tyr His Gln
        435                 440                 445
ggc ctc aag att gac cta gag acc cac tcc ttg cca gtt gtg gtg atc     1392
Gly Leu Lys Ile Asp Leu Glu Thr His Ser Leu Pro Val Val Val Ile
    450                 455                 460
tcc aac atc tgt cag atg cca aat gcc tgg gcg tcc atc ctg tgg tac     1440
Ser Asn Ile Cys Gln Met Pro Asn Ala Trp Ala Ser Ile Leu Trp Tyr
465                 470                 475                 480
aac atg ctg acc aac aat ccc aag aat gta aac ttt ttt acc aag ccc     1488
Asn Met Leu Thr Asn Asn Pro Lys Asn Val Asn Phe Phe Thr Lys Pro
                485                 490                 495
cca att gga acc tgg gat caa gtg gcc gag gtc ctg agc tgg cag ttc     1536
Pro Ile Gly Thr Trp Asp Gln Val Ala Glu Val Leu Ser Trp Gln Phe
            500                 505                 510
tcc tcc acc acc aag cga gga ctg agc atc gag cag ctg act aca ctg     1584
Ser Ser Thr Thr Lys Arg Gly Leu Ser Ile Glu Gln Leu Thr Thr Leu
        515                 520                 525
gca gag aaa ctc ttg gga cct ggt gtg aat tat tca ggg tgt cag atc     1632
Ala Glu Lys Leu Leu Gly Pro Gly Val Asn Tyr Ser Gly Cys Gln Ile
    530                 535                 540
aca tgg gct aaa ttt tgc aaa gaa aac atg gct ggc aag ggc ttc tcc     1680
Thr Trp Ala Lys Phe Cys Lys Glu Asn Met Ala Gly Lys Gly Phe Ser
545                 550                 555                 560
ttc tgg gtc tgg cta gac aat atc atc gac ctt gtg aaa aag tac atc     1728
Phe Trp Val Trp Leu Asp Asn Ile Ile Asp Leu Val Lys Lys Tyr Ile
                565                 570                 575
ctg gcc ctt tgg aac gaa ggg tac atc atg ggc ttt atc agt aag gag     1776
Leu Ala Leu Trp Asn Glu Gly Tyr Ile Met Gly Phe Ile Ser Lys Glu
            580                 585                 590
cgg gag cgg gcc atc ttg agc act aag cct cca ggc acc ttc ctg cta     1824
Arg Glu Arg Ala Ile Leu Ser Thr Lys Pro Pro Gly Thr Phe Leu Leu
        595                 600                 605
aga ttc agt gaa agc agc aaa gaa gga ggc gtc act ttc act tgg gtg     1872
Arg Phe Ser Glu Ser Ser Lys Glu Gly Gly Val Thr Phe Thr Trp Val
    610                 615                 620
gag aag gac atc agc ggt aag acc cag atc cag tcc gtg gaa cca tac     1920
Glu Lys Asp Ile Ser Gly Lys Thr Gln Ile Gln Ser Val Glu Pro Tyr
625                 630                 635                 640
aca aag cag cag ctg aac aac atg tca ttt gct gaa atc atc atg ggc     1968
Thr Lys Gln Gln Leu Asn Asn Met Ser Phe Ala Glu Ile Ile Met Gly
                645                 650                 655
tat aag atc atg gat gct acc aat atc ctg gtg tct cca ctg gtc tat     2016
Tyr Lys Ile Met Asp Ala Thr Asn Ile Leu Val Ser Pro Leu Val Tyr
            660                 665                 670
ctc tat cct gac att ccc aag gag gag gca ttc gga aag tat tgt cgg     2064
Leu Tyr Pro Asp Ile Pro Lys Glu Glu Ala Phe Gly Lys Tyr Cys Arg
        675                 680                 685
cca gag agc cag gag cat cct gaa gct gac cca ggt agc gct gcc cca     2112
Pro Glu Ser Gln Glu His Pro Glu Ala Asp Pro Gly Ser Ala Ala Pro
    690                 695                 700
tac ctg aag acc aag ttt atc tgt gtg aca cca acg acc tgc agc aat     2160
Tyr Leu Lys Thr Lys Phe Ile Cys Val Thr Pro Thr Thr Cys Ser Asn
705                 710                 715                 720
acc att gac ctg ccg atg tcc ccc cgc act tta gat tca ttg atg cag     2208
Thr Ile Asp Leu Pro Met Ser Pro Arg Thr Leu Asp Ser Leu Met Gln
                725                 730                 735
ttt gga aat aat ggt gaa ggt gct gaa ccc tca gca gga ggg cag ttt     2256
Phe Gly Asn Asn Gly Glu Gly Ala Glu Pro Ser Ala Gly Gly Gln Phe
            740                 745                 750
gag tcc ctc acc ttt gac atg gag ttg acc tcg gag tgc gct acc tcc     2304
Glu Ser Leu Thr Phe Asp Met Glu Leu Thr Ser Glu Cys Ala Thr Ser
        755                 760                 765
ccc atg tgaggagctg agaacggaag ctgcagaaag atac                       2344
Pro Met
    770
 
           
             2 
             770 
             PRT 
             Human 
           
            2
Met Ala Gln Trp Asn Gln Leu Gln Gln Leu Asp Thr Arg Tyr Leu Glu
1               5                   10                  15
Gln Leu His Gln Leu Tyr Ser Asp Ser Phe Pro Met Glu Leu Arg Gln
            20                  25                  30
Phe Leu Ala Pro Trp Ile Glu Ser Gln Asp Trp Ala Tyr Ala Ala Ser
        35                  40                  45
Lys Glu Ser His Ala Thr Leu Val Phe His Asn Leu Leu Gly Glu Ile
    50                  55                  60
Asp Gln Gln Tyr Ser Arg Phe Leu Gln Glu Ser Asn Val Leu Tyr Gln
65                  70                  75                  80
His Asn Leu Arg Arg Ile Lys Gln Phe Leu Gln Ser Arg Tyr Leu Glu
                85                  90                  95
Lys Pro Met Glu Ile Ala Arg Ile Val Ala Arg Cys Leu Trp Glu Glu
            100                 105                 110
Ser Arg Leu Leu Gln Thr Ala Ala Thr Ala Ala Gln Gln Gly Gly Gln
        115                 120                 125
Ala Asn His Pro Thr Ala Ala Val Val Thr Glu Lys Gln Gln Met Leu
    130                 135                 140
Glu Gln His Leu Gln Asp Val Arg Lys Arg Val Gln Asp Leu Glu Gln
145                 150                 155                 160
Lys Met Lys Val Val Glu Asn Leu Gln Asp Asp Phe Asp Phe Asn Tyr
                165                 170                 175
Lys Thr Leu Lys Ser Gln Gly Asp Met Gln Asp Leu Asn Gly Asn Asn
            180                 185                 190
Gln Ser Val Thr Arg Gln Lys Met Gln Gln Leu Glu Gln Met Leu Thr
        195                 200                 205
Ala Leu Asp Gln Met Arg Arg Ser Ile Val Ser Glu Leu Ala Gly Leu
    210                 215                 220
Leu Ser Ala Met Glu Tyr Val Gln Lys Thr Leu Thr Asp Glu Glu Leu
225                 230                 235                 240
Ala Asp Trp Lys Arg Arg Gln Gln Ile Ala Cys Ile Gly Gly Pro Pro
                245                 250                 255
Asn Ile Cys Leu Asp Arg Leu Glu Asn Trp Ile Thr Ser Leu Ala Glu
            260                 265                 270
Ser Gln Leu Gln Thr Arg Gln Gln Ile Lys Lys Leu Glu Glu Leu Gln
        275                 280                 285
Gln Lys Val Ser Tyr Lys Gly Asp Pro Ile Val Gln His Arg Pro Met
    290                 295                 300
Leu Glu Glu Arg Ile Val Glu Leu Phe Arg Asn Leu Met Lys Ser Ala
305                 310                 315                 320
Phe Val Val Glu Arg Gln Pro Cys Met Pro Met His Pro Asp Arg Pro
                325                 330                 335
Leu Val Ile Lys Thr Gly Val Gln Phe Thr Thr Lys Val Arg Leu Leu
            340                 345                 350
Val Lys Phe Pro Glu Leu Asn Tyr Gln Leu Lys Ile Lys Val Cys Ile
        355                 360                 365
Asp Lys Asp Ser Gly Asp Val Ala Ala Leu Arg Gly Ser Arg Lys Phe
    370                 375                 380
Asn Ile Leu Gly Thr Asn Thr Lys Val Met Asn Met Glu Glu Ser Asn
385                 390                 395                 400
Asn Gly Ser Leu Ser Ala Glu Phe Lys His Leu Thr Leu Arg Glu Gln
                405                 410                 415
Arg Cys Gly Asn Gly Gly Arg Ala Asn Cys Asp Ala Ser Leu Ile Val
            420                 425                 430
Thr Glu Glu Leu His Leu Ile Thr Phe Glu Thr Glu Val Tyr His Gln
        435                 440                 445
Gly Leu Lys Ile Asp Leu Glu Thr His Ser Leu Pro Val Val Val Ile
    450                 455                 460
Ser Asn Ile Cys Gln Met Pro Asn Ala Trp Ala Ser Ile Leu Trp Tyr
465                 470                 475                 480
Asn Met Leu Thr Asn Asn Pro Lys Asn Val Asn Phe Phe Thr Lys Pro
                485                 490                 495
Pro Ile Gly Thr Trp Asp Gln Val Ala Glu Val Leu Ser Trp Gln Phe
            500                 505                 510
Ser Ser Thr Thr Lys Arg Gly Leu Ser Ile Glu Gln Leu Thr Thr Leu
        515                 520                 525
Ala Glu Lys Leu Leu Gly Pro Gly Val Asn Tyr Ser Gly Cys Gln Ile
    530                 535                 540
Thr Trp Ala Lys Phe Cys Lys Glu Asn Met Ala Gly Lys Gly Phe Ser
545                 550                 555                 560
Phe Trp Val Trp Leu Asp Asn Ile Ile Asp Leu Val Lys Lys Tyr Ile
                565                 570                 575
Leu Ala Leu Trp Asn Glu Gly Tyr Ile Met Gly Phe Ile Ser Lys Glu
            580                 585                 590
Arg Glu Arg Ala Ile Leu Ser Thr Lys Pro Pro Gly Thr Phe Leu Leu
        595                 600                 605
Arg Phe Ser Glu Ser Ser Lys Glu Gly Gly Val Thr Phe Thr Trp Val
    610                 615                 620
Glu Lys Asp Ile Ser Gly Lys Thr Gln Ile Gln Ser Val Glu Pro Tyr
625                 630                 635                 640
Thr Lys Gln Gln Leu Asn Asn Met Ser Phe Ala Glu Ile Ile Met Gly
                645                 650                 655
Tyr Lys Ile Met Asp Ala Thr Asn Ile Leu Val Ser Pro Leu Val Tyr
            660                 665                 670
Leu Tyr Pro Asp Ile Pro Lys Glu Glu Ala Phe Gly Lys Tyr Cys Arg
        675                 680                 685
Pro Glu Ser Gln Glu His Pro Glu Ala Asp Pro Gly Ser Ala Ala Pro
    690                 695                 700
Tyr Leu Lys Thr Lys Phe Ile Cys Val Thr Pro Thr Thr Cys Ser Asn
705                 710                 715                 720
Thr Ile Asp Leu Pro Met Ser Pro Arg Thr Leu Asp Ser Leu Met Gln
                725                 730                 735
Phe Gly Asn Asn Gly Glu Gly Ala Glu Pro Ser Ala Gly Gly Gln Phe
            740                 745                 750
Glu Ser Leu Thr Phe Asp Met Glu Leu Thr Ser Glu Cys Ala Thr Ser
        755                 760                 765
Pro Met
    770
 
           
             3 
             424 
             DNA 
             Human 
             
               CDS 
               (2)..(424) 
             
             
               misc_feature 
               (2)..(424) 
               note “SH2 domain of the published hSTAT3
      sequence (Akira et al.) 
             
           
            3
c tgg cta gac aat atc atc gac ctt gtg aaa aag tat atc ttg gcc ctt     49
  Trp Leu Asp Asn Ile Ile Asp Leu Val Lys Lys Tyr Ile Leu Ala Leu
  1               5                   10                  15
tgg aat gaa ggg tac atc atg ggt ttc atc agc aag gag cgg gag cgg       97
Trp Asn Glu Gly Tyr Ile Met Gly Phe Ile Ser Lys Glu Arg Glu Arg
            20                  25                  30
gcc atc ttg agc act aag ccc cca ggc acc ttc ctg ctg cgc ttc agt      145
Ala Ile Leu Ser Thr Lys Pro Pro Gly Thr Phe Leu Leu Arg Phe Ser
        35                  40                  45
gaa agc agc aaa gaa gga ggc gtc act ttc act tgg gtg gag aag gac      193
Glu Ser Ser Lys Glu Gly Gly Val Thr Phe Thr Trp Val Glu Lys Asp
    50                  55                  60
atc agc ggt aag acc cag atc cag tcc gtg gaa cca tac aca aag cag      241
Ile Ser Gly Lys Thr Gln Ile Gln Ser Val Glu Pro Tyr Thr Lys Gln
65                  70                  75                  80
cag ctg aac aac atg tca ttt gct gaa atc atc atg ggc tat aag atc      289
Gln Leu Asn Asn Met Ser Phe Ala Glu Ile Ile Met Gly Tyr Lys Ile
                85                  90                  95
atg gat gct acc aat atc ctg ttg tct cca ctt gtc tat ctc tat cct      337
Met Asp Ala Thr Asn Ile Leu Leu Ser Pro Leu Val Tyr Leu Tyr Pro
            100                 105                 110
gac att ccc aag gag gag gca ttc ggg aag tat tgt cgg cca gag agc      385
Asp Ile Pro Lys Glu Glu Ala Phe Gly Lys Tyr Cys Arg Pro Glu Ser
        115                 120                 125
cag gag cat cct gaa gct gac cca ggt agc gct gcc cca                  424
Gln Glu His Pro Glu Ala Asp Pro Gly Ser Ala Ala Pro
    130                 135                 140
 
           
             4 
             141 
             PRT 
             Human 
             
               misc_feature 
               (2)..(424) 
               note ”SH2 domain of the published hSTAT3
      sequence (Akira et al.) 
             
           
            4
Trp Leu Asp Asn Ile Ile Asp Leu Val Lys Lys Tyr Ile Leu Ala Leu
1               5                   10                  15
Trp Asn Glu Gly Tyr Ile Met Gly Phe Ile Ser Lys Glu Arg Glu Arg
            20                  25                  30
Ala Ile Leu Ser Thr Lys Pro Pro Gly Thr Phe Leu Leu Arg Phe Ser
        35                  40                  45
Glu Ser Ser Lys Glu Gly Gly Val Thr Phe Thr Trp Val Glu Lys Asp
    50                  55                  60
Ile Ser Gly Lys Thr Gln Ile Gln Ser Val Glu Pro Tyr Thr Lys Gln
65                  70                  75                  80
Gln Leu Asn Asn Met Ser Phe Ala Glu Ile Ile Met Gly Tyr Lys Ile
                85                  90                  95
Met Asp Ala Thr Asn Ile Leu Leu Ser Pro Leu Val Tyr Leu Tyr Pro
            100                 105                 110
Asp Ile Pro Lys Glu Glu Ala Phe Gly Lys Tyr Cys Arg Pro Glu Ser
        115                 120                 125
Gln Glu His Pro Glu Ala Asp Pro Gly Ser Ala Ala Pro
    130                 135                 140
 
           
             5 
             424 
             DNA 
             Mouse 
             
               CDS 
               (2)..(424) 
             
             
               misc_feature 
               (2)..(424) 
               note “SH2 domain of murine STAT3” 
             
           
            5
c tgg cta gac aat atc atc gac ctt gtg aaa aag tat atc ttg gcc ctt     49
  Trp Leu Asp Asn Ile Ile Asp Leu Val Lys Lys Tyr Ile Leu Ala Leu
  1               5                   10                  15
tgg aat gaa ggg tac atc atg ggt ttc atc agc aag gag cgg gag cgg       97
Trp Asn Glu Gly Tyr Ile Met Gly Phe Ile Ser Lys Glu Arg Glu Arg
            20                  25                  30
gcc atc cta agc aca aag ccc ccg ggc acc ttc cta ctg cgc ttc agc      145
Ala Ile Leu Ser Thr Lys Pro Pro Gly Thr Phe Leu Leu Arg Phe Ser
        35                  40                  45
gag agc agc aaa gaa gga ggg gtc act ttc act tgg gtg gaa aag gac      193
Glu Ser Ser Lys Glu Gly Gly Val Thr Phe Thr Trp Val Glu Lys Asp
    50                  55                  60
atc agt ggc aag acc cag atc cag tct gta gag cca tac acc aag cag      241
Ile Ser Gly Lys Thr Gln Ile Gln Ser Val Glu Pro Tyr Thr Lys Gln
65                  70                  75                  80
cag ctg aac aac atg tca ttt gct gaa atc atc atg ggc tat aag atc      289
Gln Leu Asn Asn Met Ser Phe Ala Glu Ile Ile Met Gly Tyr Lys Ile
                85                  90                  95
atg gat gcg acc aac atc ctg gtg tct cca ctt gtc tac ctc tac ccc      337
Met Asp Ala Thr Asn Ile Leu Val Ser Pro Leu Val Tyr Leu Tyr Pro
            100                 105                 110
gac att ccc aag gag gag gca ttt gga aag tac tgt agg ccc gag agc      385
Asp Ile Pro Lys Glu Glu Ala Phe Gly Lys Tyr Cys Arg Pro Glu Ser
        115                 120                 125
cag gag cac ccc gaa gcc gac cca ggt agc tct gcc cca                  424
Gln Glu His Pro Glu Ala Asp Pro Gly Ser Ser Ala Pro
    130                 135                 140
 
           
             6 
             141 
             PRT 
             Mouse 
             
               misc_feature 
               (2)..(424) 
               note “SH2 domain of murine STAT3” 
             
           
            6
Trp Leu Asp Asn Ile Ile Asp Leu Val Lys Lys Tyr Ile Leu Ala Leu
1               5                   10                  15
Trp Asn Glu Gly Tyr Ile Met Gly Phe Ile Ser Lys Glu Arg Glu Arg
            20                  25                  30
Ala Ile Leu Ser Thr Lys Pro Pro Gly Thr Phe Leu Leu Arg Phe Ser
        35                  40                  45
Glu Ser Ser Lys Glu Gly Gly Val Thr Phe Thr Trp Val Glu Lys Asp
    50                  55                  60
Ile Ser Gly Lys Thr Gln Ile Gln Ser Val Glu Pro Tyr Thr Lys Gln
65                  70                  75                  80
Gln Leu Asn Asn Met Ser Phe Ala Glu Ile Ile Met Gly Tyr Lys Ile
                85                  90                  95
Met Asp Ala Thr Asn Ile Leu Val Ser Pro Leu Val Tyr Leu Tyr Pro
            100                 105                 110
Asp Ile Pro Lys Glu Glu Ala Phe Gly Lys Tyr Cys Arg Pro Glu Ser
        115                 120                 125
Gln Glu His Pro Glu Ala Asp Pro Gly Ser Ser Ala Pro
    130                 135                 140
 
           
             7 
             47 
             DNA 
             Artificial Sequence 
             
                 
             
           
            7
aacaccatgg cctggctaga caatatcatc gaccttgtga aaaagta                   47
 
           
             8 
             39 
             DNA 
             Artificial Sequence 
             
                 
             
           
            8
atatatggat cctggggcag cgctacctgg gtcagcttc                            39
 
           
             9 
             35 
             DNA 
             Artificial Sequence 
             
                 
             
           
            9
tccccggaag cttcacacgc gcagccccgg cttct                                35
 
           
             10 
             30 
             DNA 
             Artificial Sequence 
             
                 
             
           
            10
gttcatcact tttgtgtttg tgcccagaat                                      30
 
           
             11 
             30 
             DNA 
             Artificial Sequence 
             
                 
             
           
            11
gacaaagact ctggggacgt tgcagctctc                                      30
 
           
             12 
             35 
             DNA 
             Artificial Sequence 
             
                 
             
           
            12
tcagtcctcg agtatctttc tgcagcttcc gttct                                35
 
           
             13 
             22 
             DNA 
             Artificial Sequence 
             
                 
             
           
            13
tgaagggtac atcatgggtt tc                                              22
 
           
             14 
             29 
             DNA 
             Artificial Sequence 
             
                 
             
           
            14
tcaggataga gatagacaag tggagacaa                                       29
 
           
             15 
             27 
             DNA 
             Artificial Sequence 
             
                 
             
           
            15
cctccttctt tgctgctttc actgaag                                         27
 
           
             16 
             22 
             DNA 
             Artificial Sequence 
             
                 
             
           
            16
cgaagggtac atcatgggct tt                                              22
 
           
             17 
             30 
             DNA 
             Artificial Sequence 
             
                 
             
           
            17
cctccttctt tgctgctttc actgaatctt                                      30
 
           
             18 
             33 
             DNA 
             Artificial Sequence 
             
                 
             
           
            18
tgaagggtac atcatgggtt tcatcagtaa gga                                  33
 
           
             19 
             37 
             DNA 
             Artificial Sequence 
             
                 
             
           
            19
tcaggataga gatagacaag tggagacaac aggatat                              37