Abstract:
The invention relates to new members of the GAGE family referred to as GAGE-7B and GAGE-8. There are differences between these two molecules and the previously described members of the GAGE family on the genomic DNA, complementary DNA, and amino acid level.

Description:
FIELD OF THE INVENTION 
     This invention relates to a nucleic acid molecule which codes for a tumor rejection antigen precursor. More particularly, the invention concerns genes, whose tumor rejection antigen precursor is processed, inter alia, into at least one tumor rejection antigen that is presented by MHC molecules. The genes in question are members of the GAGE family of genes. 
     BACKGROUND AND PRIOR ART 
     The process by which the mammalian immune system recognizes and reacts to foreign or alien materials is a complex one. An important facet of the system is the T lymphocyte, or “T cell” response. This response requires that T cells recognize and interact with complexes of cell surface molecules, referred to as human leukocyte antigens (“HLA”), or major histocompatibility complexes (“MHCs”), and peptides. The peptides are derived from larger molecules which are processed by the cells which also present the HLA/MHC molecule. See in this regard Male et al.,  Advanced Immunology  (J. P. Lipincott Company, 1987), especially chapters 6-10. The interaction of T cells and HLA/peptide complexes is restricted, requiring a T cell specific for a particular combination of an HLA molecule and a peptide. If a specific T cell is not present, there is no T cell response even if its partner complex is present. Similarly, there is no response if the specific complex is absent, but the T cell is present. This mechanism is involved in the immune system&#39;s response to foreign materials, in autoimmune pathologies, and in responses to cellular abnormalities. Much work has focused on the mechanisms by which proteins are processed into the HLA binding peptides. See, in this regard, Barinaga, Science 257: 880 (1992); Fremont et al., Science 257: 919 (1992); Matsumura et al., Science 257: 927 (1992); Latron et al., Science 257: 964 (1992). Also see Engelhard, Ann. Rev. Immunol. 12: 181-207 (1994). 
     The mechanism by which T cells recognize cellular abnormalities has also been implicated in cancer. For example, in PCT application PCT/US92/04354, filed May 22, 1992, published on Nov. 26, 1992, and incorporated by reference, a family of genes is disclosed, which are processed into peptides which, in turn, are expressed on cell surfaces, which can lead to lysis of the tumor cells by specific CTLs cytolytic T lymphocytes, or “CTLs” hereafter. The genes are said to code for “tumor rejection antigen precursors” or “TRAP” molecules, and the peptides derived therefrom are referred to as “tumor rejection antigens” or “TRAs”. See Traversari et al., Immunogenetics 35: 145 (1992); van der Bruggen et al., Science 254: 1643 (1991), for further information on this family of genes. Also, see U.S. Pat. No. 5,342,774. 
     In U.S. Pat. No. 5,405,940, the disclosure of which is incorporated by reference, it is explained that the MAGE genes code for proteins which are processed to nonapeptides which are then presented by an HLA-A1 molecule. The reference teaches that given the known specificity of particular peptides for particular HLA molecules, one should expect a particular peptide to preferably bind to one HLA molecule. This is important, because different individuals possess different HLA phenotypes. As a result, while identification of a particular peptide as being a partner for a specific HLA molecule has diagnostic and therapeutic ramifications, these are only relevant for individuals with that particular HLA phenotype. There is a need for further work in the area, because cellular abnormalities are not restricted to one particular HLA phenotype, and targeted therapy requires some knowledge of the phenotype of the abnormal cells at issue. 
     In U.S. Pat. No. 5,629,166 incorporated by reference, the fact that the MAGE-1 expression product is processed to a second TRA is disclosed. This second TRA is presented by HLA-C clone 10 molecules. The disclosure shows that a given TRAP can yield a plurality of TRAs. Also, see U.S. Pat. No. 5,554,506, incorporated by reference, teaching peptides which bind to HLA-A2. 
     U.S. Pat. Nos. 5,530,096 and 5,487,934 incorporated by reference herein teach that tyrosinase, a molecule which is produced by some normal cells (e.g., melanocytes), is processed in tumor cells to yield peptides presented by HLA-A2 molecules. 
     In U.S. Pat. No 5,620,886, incorporated by reference in its entirety, a second TRA, not derived from tyrosinase is taught to be presented by HLA-A2 molecules. The TRA is derived from a TRAP, but is coded for by a non-MAGE gene. This disclosure shows that a particular HLA molecule may present TRAs derived from different sources. 
     In U.S. Pat. No. 5,571,711, filed Jun. 17, 1993 and incorporated by reference herein, an unrelated tumor rejection antigen precursor, the so-called “BAGE” precursor, is described. The BAGE precursor is not related to the MAGE family. 
     A further family of genes which are processed into tumor rejection antigens is taught by U.S. Pat. Nos. 5,610,013 and 5,648,226, as well as patent applications Ser. Nos. 08/531,662 and 08/602,039, filed on Sep. 21, 1995 and Feb. 15, 1996 respectively, both of which have been allowed, and U.S. patent applications Ser. No. 08/669,161 and 09/012,818, filed on Jun. 24, 1996 and Jan. 23, 1998, respectively. All of these applications are incorporated by reference. They reveal that there is a family of genes, the “GAGE” genes, which are related to each other. Six members of the GAGE family are described in these references. 
     It has now been found that there are at least two further members of the GAGE family, referred to hereafter as GAGE-7 and GAGE-8. These genes, as well as other aspects of the inventions, will be described in detail in the disclosure which follows. 
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
     EXAMPLE 1 
       
     Melanoma cell line MZ2-MEL and cell lines derived therefrom are known. See, e.g., U.S. Pat. No. 5,342,774, incorporated by reference. One subdlone, i.e., MZ2-MEL 3.0 was obtained by limiting dilution, and is described in the &#39;774 patent. A subline, i.e., MZ2-MEL. 43 was derived by limiting dilution of MZ2-MEL 3.0 cells which had survived mutagen treatment. See Herin, et al, Int. J. Canc. 39:390-396 (1987); Van den Eynde, et al, Int J. Canc. 44:634-640 (1980). This subline had been used as a source of cDNA from which nucleic acid molecules encoding GAGE 1-6 were isolated. See U.S. Pat. Nos. 5,610,013; 5,648,226; application Ser. Nos. 08/531,662; 08/602,039; 08/669,661; and 09/012,818 cited supra, and Van den Eynde, et al, J. Exp. Med. 182:689-698 (1995), all of which are incorporated by reference. 
     The cDNA library from MZ2-MEL.43 was rescreened, using the same protocols as are set forth in the above referenced patents and 1995 paper. Two additional positive clones were identified. These molecules were named GAGE-7B and GAGE-8. They are discussed further, infra. The nucleotide sequences for cDNA for these molecules are set forth as SEQ ID NO: 1 (GAGE-8), and SEQ ID NOS: 2 and 3 (GAGE-7B). 
     EXAMPLE 2 
     These experiments describe the isolation of genomic DNA molecules encoding GAGE-7B. 
     Peripheral blood lymphocytes (PBLs) were isolated, and grown, using standard methodologies. The genomic DNA was then isolated from the PBLs, partially digested with endonuclease Sau3A1, size fractionated using NaCl density gradient centrifugation, and then ligated into GEM-11 cloning vector, which had been digested with BamHI and EcoRI. 
     The phage library was screened, using a probe labeled with α 32 P dCTP, consisting of nucleotides 18-309 of cDNA for GAGE-1. Conditions for this Southern hybridization was standard, as described by Sambrook et al: Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 1989), incorporated by reference. The washing conditions were 0.2×SSC, 0.1% SDS, at  65° C.    
     One of the positive clones was analyzed, and found to contain an insert corresponding to GAGE-7B. The sequence, set forth at SEQ ID NO:3, contains 5 exons, including an open reading frame over exons 2 to 5, which encodes a 117 amino acid product. 
     The fourth intron of this sequence includes two regions which show strong homology with a region found only in GAGE-1. There is a 561 base pair segment positioned in between these regions at nucleotides 7109-7659, which corresponds to a truncated, L1 retroposon which belongs to the family of long interspersed repeated elements, or “LINE”; as described by Hutchinson, et al, in Berg, et al, eds, “Mobile DNA” (Am. Soc. Microbiol. 1989), incorporated by reference. The LINE element is flanked by a perfect 13 base pair target site duplication, and contains part of the reverse transcriptase coding region, the 3′-untranslated region, and the poly-A tail of the original retroposon. 
     EXAMPLE 3 
     A cosmid library was prepared using genomic DNA from renal cell carcinoma cell line LE9211-RCC, following the methodologies described by Lurquin, et al, Cell 58: 293-303 (1989), and screened using the Southern hybridization method set forth in example 2, using the same probe. 
     A cosmid was identified which contained genomic DNA for GAGE-8. Its structure was the same as that of GAGE-7B, including the LINE insertion discussed supra. 
     EXAMPLE 4 
     These experiments describe how the chromosomal location of the GAGE genes was determined. Southern blot analysis was carried out on a panel of hamster or mouse x human somatic cell hybrids, obtained from the Human Genetic Mutant Cell Repository. The DNA from these somatic cell hybrids was isolated, digested with EcoRI, and used to prepare Southern blots, in accordance with Arden, at el, Cytogenet. Cell Genet. 53:161-165 (1990), incorporated by reference. The GAGE-1 probe, labeled with β 32 P dCTP, as described supra, was used. A single, EcoRI band of 4.3 kilobases was detected, indicating that the EcoRI sites defining the fragment are conserved in all GAGE genes. The only hybridization signal came from a hybrid containing the human X chromosome. No signal came from hybrids containing human autosomes, or the Y chromosome. 
     Experiments were than carried out to refine the localization of the GAGE locus. Somatic cell hybrids containing only a portion of the X chromosome were analyzed via Southern hybridization, as described supra, as well as by PCR. 
     For PCR, primers corresponding to nucleotides 453-470 of GAGE-1 cDNA (sense), and nucleotides 613-630 of GAGE-1 cDNA (antisense), were used. These should amplify a 0.7 kb fragment of genomic DNA, and a fragment consisting of nucleotides 453-630 of GAGE-1 cDNA, as set forth in U.S. Pat. No. 5,610,013 at SEQ ID NO: 1. Thirty five cycles of amplification were carried out, each cycle consisting of denaturation at 94° C. (1 minute), annealing at 50° C. (1 minute), and extension at 72° C. for 1 minute. The PCR was preceded by 3 minutes of incubation at 94° C., and was followed by a soak at 72° C. for 10 minutes. Amplified products were electrophoresed on 2% agarose gels, and were visualized by ethidium bromide staining. The analysis revealed that the GAGE genes are located in chromosomal region Xp21-Xq13. 
     EXAMPLE 5 
     A further set of experiments were carried out to find the location of the GAGE locus, using fluorescence in situ hybridization, or “FISH”. To accomplish this, PBLs were stimulated with PHA, and cultured for 72 hours. Banded chromosomes were obtained by inoculating some cultures with 5-bromodeoxyuridine, in accordance with Lemieux, et al, Cytogenet. Cell Genet 59:311-312 (1992). Cytogenetic harvests, and slide preparations were prepared using standard methods. Slides were stored at −80° C. until used. 
     FISH hybridization to metaphase chromosomes was carried out following Pinkel, et al, Proc. Natl. Acad. Sci USA 83:2934-2938 (1986). Briefly, slides were denatured for 2 minutes in 70% formamide/2×SSC (pH 7.0), and then dehydrated in ice cold ethanol. A cosmid which contained gDNA for GAGE-7B was used as a probe. The probe (100 ng) was labeled with digoxigenin, preannealed with 100 mg of COT-1 DNA, dissolved in buffer (50% formamide, 2×SSC), denatured at 75° C. for 5 minutes, and then applied to slides. The probes were hybridized to the material on the slides, overnight at 37° C., in a humid chamber. 
     After the incubation, the slides were washed using standard procedures, and then analyzed using standard FITC-digoxigenin detection methods, together with an amplification protocol for dual color FISH. The slides were counterstained by mounting in an antifade solution containing 1 mg/ml phenylenediamine and 0.3 mg/ml propidium iodide. Spreads were examined, and photographed. A signal was deemed to be specific only if detected on each chromatid of a single chromosome. Chromosome identification was performed via simultaneous hybridization with the satellite repeat probe, or by R-banding, using 5-bromodeoxyuridine in accordance with Lemieux, et at, supra. 
     These experiments indicated that the GAGE locus is in the p11.2-p11.4 region of the X chromosome. 
     EXAMPLE 6 
     These experiments were designed to determine expression of GAGE genes in various cell and tumor types. For each type of cell assayed, total RNA was extracted, using standard guanidium—isothiocyanate procedures, as taught by e.g., Davis, et al. in Basic Methods In Molecular Biology, Elsevier Science Publishing Co., N.Y. (1986), pp. 130-135. Reverse transcription was carried out on 2 ug samples of the total RNA, using 2 mM of oligo(dT) 15  primer, in a reaction volume of 20 ul. Portions of the resulting cDNA (1/20 of the product), were used in the PCR amplification. In order to amplify GAGE-1, 2, and 8, the primers used were: 
     5′-GACCAAGACG CTACGTAG-3′ 
     (sense, SEQ ID NO: 4) and 
     5′-CCATCAGGAC CATCTTCA- 3′ 
     (antisense, SEQ ID NO: 5) 
     For GAGE-3, 4, 5, 6 &amp; 7B, the primers were: 
     5′-GACCAAGGCG CTATGTAC-3′ 
     (sense, SEQ ID NO: 6) 
     and SEQ ID NO: 5 
     For all amplifications, the denaturation step was 94° C. for 5 minutes, then 30 cycles of amplification (1 minute at 94° C., 2 minutes at 58° C., 2 minutes at 72° C.), then a final extension step of 72° C. for 15 minutes. The products were analyzed by agarose gel electrophoresis, with RNA integrity being checked by reverse transcription and amplification of β-actin mRNA. 
     When these primers are used, SEQ ID NOS: 4 and 5 produce a fragment consisting of nucleotides 107-350 of SEQ ID NO: 1. SEQ ID NOS: 5 and 6 produce a fragment consisting of nucleotides 92-335 of SEQ ED NO: 2. 
     Table 1, which follows, shows the results. The highest fraction of positive tumors were found in melanoma, esophageal and lung carcinomas. GAGE 1, 2 and 8 was found in prostate carcinomas, breast carcinomas, and sarcomas. GAGE 3, 4, 5, 6 and 7B were not found in this tumors. No expression of GAGE was found in colorectal and renal carcinoma. 
     
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
             
             
               
                 Expression of the GAGE genes in tumors 
               
             
          
           
               
                   
                 Number of samples 
                   
               
               
                   
                 ALL GAGE 
               
             
          
           
               
                   
                   
                   
                   
                 1, 2, 8 
                 3-6 + 7B 
                 None 
                 % of samples 
               
               
                   
                   
                 Expression of GAGE-1, 2, 8* 
                 + 
                 + 
                 − 
                 − 
                 expressing GAGE-1, 2, 7 
               
               
                 Tumor 
                 tested 
                 Expression of GAGE-3, 4, 5, 6, 7B 
                 + 
                 − 
                 + 
                 − 
                 and/or GAGE-3, 4, 5, 6, 8 
               
               
                   
               
               
                   Cutaneous melanoma  (primaries) 
                 79 
                   
                 22 
                 1 
                 10 
                 46 
                    42% 
               
               
                   Cutaneous melanoma  (metastases) 
                 211 
                   
                 79 
                 8 
                 26 
                 98 
                    54% 
               
               
                   Esophageal squamous  cell carcinoma 
                 18 
                   
                 7 
                 1 
                 1 
                 9 
                    50% 
               
               
                 
                   Esophageal adenocarcinoma 
                 
                 5 
                   
                 1 
                 0 
                 0 
                 4 
                 20 
               
               
                 Lung squamous cell carcinoma 
                 83 
                   
                 28 
                 4 
                 7 
                 44 
                 47 
               
               
                 Lung adenocarcinoma 
                 42 
                   
                 13 
                 2 
                 6 
                 21 
                 50 
               
               
                 Head &amp; neck carcinoma 
                 92 
                   
                 21 
                 3 
                 5 
                 63 
                 31 
               
               
                 Bladder carcinoma (superficial) 
                 35 
                   
                 1 
                 0 
                 0 
                 34 
                  3 
               
               
                 Bladder carcinoma (infiltrating) 
                 40 
                   
                 8 
                 2 
                 6 
                 24 
                 40 
               
               
                 Leukemia 
                 76 
                   
                 0 
                 0 
                 3 
                 73 
                  4 
               
               
                   
               
             
          
         
       
     
     EXAMPLE 7 
     In order to deterrnine if expression of GAGE genes could be induced by demethylation, samples of cultured tumor and normal cells were incubated for 72 hours in culture medium containing 1 uM 5-aza-2′-deoxycytidine. SEQ ID NOS: 4 and 5, supra, were used in the amplification protocol. GAGE 1, 2, and 8 were found to have been induced in sarcoma and melanoma cell lines. All GAGE genes were found to be expressed following treatment of PHA stimulated PBLs. 
     The foregoing examples set forth the invention, which includes isolated nucleic acid molecules which encode proteins GAGE 7B and GAGE 8. These may be, e.g., those set forth at SEQ ID NO: 1, 2 or 3, as well as all nucleic acid molecules which encode the proteins encoded by theses sequences. When GAGE-7B and GAGE-8 are compared to the other members of the GAGE family, cDNA for GAGE-8 is found to be identical to cDNA for GAGE-2 but for a single nucleotide, at nucleotide 268 (“C” in GAGE-2, versus “G” in GAGE-8). This leads to a change in the amino acid at position 74 (His in GAGE-2, Asp in GAGE-8). GAGE-7B is identical to GAGE-4, but for two nucleotides at positions 268 and 548. This first difference (“G” in GAGE-4, “C” in GAGE-8), results in a change at amino acid 74 as well (Asp in GAGE-4, His in GAGE-7B). 
     There are further differences in the organization of the genomic DNA, as explained supra. Specifically, GAGE-8 and GAGE-7B differ from GAGEs 2-6 in that they contain two inserts in the fourth intron. These inserts are found in GAGE-1 genomic DNA; however, GAGE-8 and 7B also contain a 561 base pair insert positioned in between these two inserts, which is not found in the genomic DNA of GAGE-1. 
     In addition to the nucleic acid molecules discussed supra, other features of the invention include expression vectors which include the nucleic acid molecules of the invention, operably linked to a promoter. Both cDNA and genomic DNA can be used, in expression vectors of various types. These, as well as the isolated nucleic acid molecules of the invention, can be used to make recombinant eukaryotic and prokaryotic cells, which contain either the isolated nucleic acid molecules or the expression vectors of the invention. The choice of which nucleic acid molecule or which expression vector to use will be up to the skilled artisan, depending upon the application of interest. 
     The nucleic acid molecules of the invention do include segments which correspond to peptides presented by HLA-Cw6 and HLA-A29, i.e., YRPRPRRY (SEQ ID NO:26) (GAGE 1, 2 and 8), and YYWPRPRRY (SEQ ID NO:27) (GAGE 3, 4, 5, 6 and 7B). Hence, a further aspect of the invention are recombinant cells which, in addition to including molecules which encode GAGE-7B and GAGE-8, also include one or more nucleic acid molecules which encode MHC molecules, such as HLA-Cw6 and/or HLA-A29. It is to be understood that additional genes which are processed to presented antigens may be used as well the GAGE 7B and 8 genes. 
     Also a feature of the invention are the proteins encoded by the nucleic acid molecules of the invention. As explained, supra, these proteins are similar, but not identical to other GAGE proteins. Also, part of the invention are fragments of the proteins of the invention. In particular, these fragments compare at least the first 74 amino acids encoded by the SEQ ID NO: 1, 2 or 3, and no more than the entire molecule encoded by these sequences. These proteins are set forth at SEQ ID NOS.:7 and 8. Also a part of the invention are those peptides, derived form GAGE 7B and/or GAGE 8, which complex to MHC molecules, thereby identify a particular molecule, and also in at least some cases, facilitating the proliferation of cytolytic T cells which recognize complexes of the peptide and the MEC molecule to which it binds. One or more of these peptides can be combined in compositions, which may also include one or more adjuvants, such as GM-CSF, an interleukin, an emulsifying oil such as Vitamin E, a saponin, etc. 
     “Minigenes” can also be produced which are nucleic acid molecules that consent of nucleotides that encode these peptides. Constructs can also be prepared, such as expression vectors, which encode one or more of these peptides. 
     An exemplary list of such peptides, with the partner MHC molecule, follows. The positions for GAGE 7B are by reference to SEQ ID NO: 7, and for GAGE 8, they are by reference to SEQ ID NO: 8 
     
       
         
               
             
               
               
               
             
           
               
                   
               
               
                 GAGE 78 
               
             
          
           
               
                 Position 
                 Sequence 
                 HLA Molecule 
               
               
                   
               
               
                 43-51 
                 EGEPATQRQ 
                 A1 
               
               
                  9-17 
                 YYWPRPRRY 
                 A24 
               
               
                 16-24 
                 RYVQPPEMI 
                 A24 
               
               
                 24-32 
                 IGPMRPEQF 
                 A24 
               
               
                 11-19 
                 WPRPRRYVQ 
                 B7 
               
               
                 19-27 
                 QPPEMIGPM 
                 B7 
               
               
                 11-19 
                 WPRPRRYVQ 
                 B8 
               
               
                 1-9 
                 MSWRGRSTY 
                 B3501 
               
               
                 19-27 
                 QPPEMIGPM 
                 B3501 
               
               
                 28-36 
                 RPEQFSDEV 
                 B3501 
               
               
                 1-9 
                 MSWRGRSTY 
                 B4403 
               
               
                 33-41 
                 DEVEPATPE 
                 B4403 
               
               
                 56-64 
                 QEGEDEGAS 
                 B4403 
               
               
                 108-116 
                 EEGEKQSQC 
                 B4403 
               
               
                 16-24 
                 RYVQPPEMI 
                 B5201 
               
               
                 19-27 
                 QPPEMIGPM 
                 B5201 
               
               
                 24-32 
                 IGPMRPEQF 
                 B5201 
               
               
                 28-36 
                 RPEQFSDEV 
                 B5201 
               
               
                  97-105 
                 MDPPNPEEV 
                 B5201 
               
               
                 19-27 
                 QPPEMIGPM 
                 Cw0602 
               
               
                 28-36 
                 RPEQFSDEV 
                 Cw0602 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
           
               
                   
               
               
                 GAGE 8 
               
             
          
           
               
                 Position 
                 Sequence 
                 HLA Molecule 
               
               
                   
               
               
                 16-24 
                 YVEPPEMIG 
                 A1 
               
               
                 42-50 
                 EGEPATQRQ 
                 A1 
               
               
                  8-16 
                 TYRPRPRRY 
                 A24 
               
               
                 15-23 
                 RYVEPPEMI 
                 A24 
               
               
                 23-31 
                 IGPMRPEQF 
                 A24 
               
               
                 10-18 
                 RPRPRRYVE 
                 B7 
               
               
                 18-26 
                 EPPEMIGPM 
                 B7 
               
               
                 1-9 
                 MSWRGRSTY 
                 B3501 
               
               
                 18-26 
                 EPPEMIGPM 
                 B3501 
               
               
                 27-35 
                 RPEQFSDEV 
                 B3501 
               
               
                 1-9 
                 MSWRGRSTY 
                 B4403 
               
               
                 33-41 
                 DEVEPATPE 
                 B4403 
               
               
                 56-64 
                 QEGEDEGAS 
                 B4403 
               
               
                 108-116 
                 EEGEKQSQC 
                 B4403 
               
               
                 18-26 
                 EPPEMIGPM 
                 Cw0602 
               
               
                 27-35 
                 RPEQFSDEV 
                 Cw0602 
               
               
                   
               
             
          
         
       
     
     Other features of the invention will be clear to the skilled artisan, and will not be set forth here. 
     The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention. 
     
       
         
           
             27 
           
           
             1 
             528 
             DNA 
             Homo sapiens 
             
 
           
            1
ctgtgaggca gtgctgtgtg gttcctgccg tccggactct ttttcctcta ctgagattca     60
tctgtgtgaa atatgagttg gcgaggaaga tcgacctatc ggcctagacc aagacgctac    120
gtagagcctc ctgaaatgat tgggcctatg cggcccgagc agttcagtga tgaagtggaa    180
ccagcaacac ctgaagaagg ggaaccagca actcaacgtc aggatcctgc agctgctcag    240
gagggagagg atgagggagc atctgcaggt caagggccga agcctgaagc tgatagccag    300
gaacagggtc acccacagac tgggtgtgag tgtgaagatg gtcctgatgg gcaggagatg    360
gacccgccaa atccagagga ggtgaaaacg cctgaagaag gtgaaaagca atcacagtgt    420
taaaagaaga cacgttgaaa tgatgcaggc tgctcctatg ttggaaattt gttcattaaa    480
attctcccaa taaagcttta cagccttctg caaagaaaaa aaaaaaaa                 528
 
           
             2 
             526 
             DNA 
             Homo sapiens 
             
 
           
            2
tggttcctgc cgtccggact ctttttcctc tactgagatt catctgtgtg aaatatgagt     60
tggcgaggaa gatcgaccta ttattggcct agaccaaggc gctatgtaca gcctcctgaa    120
atgattgggc ctatgcggcc cgagcagttc agtgatgaag tggaaccagc aacacctgaa    180
gaaggggaac cagcaactca acgtcaggat cctgcagctg ctcaggaggg agaggatgag    240
ggagcatctg caggtcaagg gccgaagcct gaagctcata gccaggaaca gggtcaccca    300
cagactgggt gtgagtgtga agatggtcct gatgggcagg agatggaccc gccaaatcca    360
gaggaggtga aaacgcctga agaaggtgaa aagcaatcac agtgttaaaa gaaggcacgt    420
tgaaatgatg caggctgctc ctatgttgga aatttgttca ttaaaattct cccaataaag    480
ctttacagcc ttctgcaaag aaaaaaaaaa aaaaaaaaaa aaaaaa                   526
 
           
             3 
             9531 
             DNA 
             Homo sapiens 
             
               unsure 
               92,232,1041,7412,9038-9040 
               identity of several nucleotides not known 
             
           
            3
gagctcgctg cagccttgac ctcctgggct caagcgctcc tcccacctca gcctcctgag     60
tagctgtgag tataggtaca tgccaccatg cncagctaat ttttcgatgg tttttttgtt    120
tgttttttgt agtgatgaga ttttctgatg ttgcttaggc tggtctcgaa gtcctgagct    180
caggtgatct ggccagctca gcctcccaaa atactaggat tacaggcgtg anttggcctg    240
gtctggtttt tcttatatag gggtcttatc tatataaaga ctaaagttaa tctgtgcctt    300
tgtgcgggtg ggctaagagc atgatgactt ttatcattct attgatttaa agaaaactgt    360
ccttgactta ccagtgtgta agtccatgaa agcataattc tgttgaaagc atatattgtt    420
aatgggtgtt gggaaccgtg cactttccgc tgctgtggga gcatgtcctt ggaggtacct    480
ttcatctgtt ttctcaactc caaacatctt aggaccatgg gttgtgactg gtaggactat    540
gtatcttgct gctttcaaga cggagtatat tttcacgtgg tgtcactctg gctgtcctgt    600
ttccctaata ctgtcacttc accctctgcg attctgatgc tacaaatgat agatatcgtt    660
ttagcatttt cttacgggtc ctagcgattc tattcatttt tctttcagtc tctttctctg    720
acttgttcac attgaacaat ttccttttgg gataggttgc tatttctgtt ttcgcaggtg    780
gtttacctgt cttcccagcc agtcacagtg gtccttgtcc ccatggtggg tccggggcaa    840
gagagggccc tgggttgggg gtggggttca gttgaagatg gggtgagttt tgaggggagc    900
actacttgag tcccagaggc ataggaaaca gcagagggag gtgggattcc cttatcctca    960
atgaggatgg gcatggaggg tttggggcgt ggcgctggga acggcagccc tccccagccc   1020
acagccgcgc atgctccctg ntcccgcctc agtgcgcatg ttcactgggc gtcttctgcc   1080
cggccccttc gcccacgtga agaacgccag ggagctgtga ggcagtgctg tgtggttcct   1140
gccgtccgga ctctttttcc tctactgaga ttcatctggt aggtgtgcag gccagtcatc   1200
ccgggggctg aagtgtgagt gagggtggag agggcctcgg gtgggtcagg cgggtccgtt   1260
cctggtctgt ggcctccgag ggagaagggc cacgaggtta cgtacctcct tacccttcac   1320
aggctgcgag gccaccggcg gcttcgtggt cgtgaagggg cctggacggg gaggaaggtg   1380
ggccgtggag gggaggctgt caggggctca ggtgaagacg gggtgagtgc tgttgggggg   1440
atggaagtcc cgaggtgccg ggatccccga cgacacaggg cagattccct gaatgggccc   1500
ggcgggggcg aggcgggcgg tgaagaaggg gcctggcacc tgggaaggct gcggcctggc   1560
gagcgccccc cccagcggtg tggagtgcgg agcgcccgag tgagaagcac tgcaaggtct   1620
cacctccgcc atggaaggtc cgaaaacagt gggaaggagt gggcgaggca gtgcggtcca   1680
accaaacttg ttgtgagggg gggtgaatgg ctctaggaag tgggagtgtg cccaaagcag   1740
caatcacgag aattgtgatt cactagggtt ttcgtgggga gtgcacttgt gaaactaaac   1800
ctcatcagaa atgacctctg tctgcggggc gcagtggcgc tcgcctacgt agtcccagtt   1860
actggggaca ctgaggtggg aggatccctt gagcgggagg tcgaggctgc agtgagctgt   1920
gatcacgccg ctgcactcca gcctgagcaa cacagcgata ccgcgtgtcc aaaagaaatt   1980
tagaaaaaaa tgtcctctgc cttttgccac acgccttaag atgattgctc tgccagcctg   2040
gccagcagaa gtggctttgt aggcactcag acagcgtaca cacgtatgct taactctggg   2100
acttattttg agagtatttt caaaagtaaa acggcaagtt aacatttatc catggaagtg   2160
atcgaatata gcagccctgt ggagcgcacg ttcccaatca cggttgtctg ttttcagtgt   2220
gaaatatgag ttggcgagga agatcgacct attattggcc tagaccaagg cgctatgtac   2280
agcctcctga aatgattggg cctatgcggg tgagtgctta aacgttaatt cgatgttttc   2340
tattagtaga aattaatttt tgtgatagcg tcgttgcatt agtgtggaaa tgctgataaa   2400
ggtctttcct gctcataaaa aatgaggatg gcatctcatg aaggaaacat tgattctgga   2460
ggattttttt ttttcctctc gtgttcttca gcttttgccc atgacttctt tctccggctt   2520
tgtttgttaa tgacagattg tacacatgta ttccaacaca gagtataata gcccccaaag   2580
tcctcgtgcg tcacttttct cacagtaacc tccctgtggg tggagtaacc ttattgggca   2640
tagagcatag agttggagaa atgtctttag gcttagttag gaccagaaat agctatgtat   2700
tctgtgtata tatgtaaaat tttgtatcaa taacgaaact tattttttat ttgcacaccc   2760
acacgtattc cccagcccga gcagttcagt gatgaagtgg aaccagcaac acctgaagaa   2820
ggggaaccag caactcaacg tcaggatcct gcagctgctc aggagggaga ggatgaggga   2880
gcatctgcag gtcaaggtga gggaaaggga agaagaacgt ctgctggtgt gtgcgtgtgt   2940
gtgtgttcgt gtgtgtgtgt gcacgtgtgt gtgtgttagg cattgtcaca taggaggaag   3000
aggaggaaag aaaacaatgg aaagaatgcc tgaaattgac tggaaaagcg aggaggctat   3060
gtagtttgca gcttagctta ggcaaatccc tcactatgat aaaagttctc gactttatga   3120
atgagagaat ggaggtgcca ggattgtgtg ttatccaaga acccttgact ggtgaataca   3180
acatttgtac tgtgttctaa ggtttgtgtc ttcctatcat gtatgttgct ggaaagaagg   3240
aagtgatttt gctgaaaatg cttaaaactc aaaaggcttt actgtaaggt agcttagtac   3300
tgacccaaga atagacccag ttcagaggag caggagcagc tccaaaaacc gagtcgctga   3360
atgttggccc ccgtttcctt tgattgatat ttttatatgg tacgtttgat aaaagctgga   3420
taaatgagga tactgccata caggtagctg gtttagtgat ttttctcagc ggcctttagg   3480
aggtgattaa atccttttat ggttagaaaa gcaaaaacgg aattatcctg agattaacgt   3540
gagatggaaa taatttctcc gagataaaat gttttgaaag gaagcattta tgtaacggag   3600
gtcatggatt attccaggga tgcactgtta aaagttccta gaatctgact gacaacaatg   3660
cccattaatt gctgtccgcc cactccctta ttctcagtgc ggggacagta tattttctgt   3720
gattcacaaa caatgttata tttggtgctt tgttcttcac ggggttcatt tatggaatat   3780
tacctttagg accttcggac ctaaatataa ctttatttga acaaagtgaa gtttctcttt   3840
accccgatag gtaatgggtg tcgtgactgt aagatttcca tagtcctcaa atccatccag   3900
ctaatcaatc cttcagaaac tgacattgta attgtaactg aaatcctacc cacgtggtag   3960
acttcagatt tctcagctga cacacactgc tgttggtact ctagggctga atataagcat   4020
tatacatgtc ctgtggttta tccttagatt gtcatttagg agaaaggtct aaagctgggc   4080
tgaatgccat gcactcatag tcccagctac ttgggaggcc gaggtgagag gattgcttga   4140
gtcctggagt tcaagcccag cctgggaaac acagtgagac ctcattgcta ataaataaat   4200
aaatgaataa ataaataaac acataaataa attcattaaa taaataaagt tttcatggta   4260
taggaaaaca cagatgcaaa gtttttgtgc ctagtggctg gtaatgttgc aaacgtaact   4320
ccttagtgaa ctgtaccact ttagttaaga tggtaaattt taggatatct gtatttttta   4380
ccacaattgg aaattccttt cttcctaaag ttcagtgcag ttatcatata ttcttttaaa   4440
tttttactgt atgtatcttc aagacataac attcatagaa aatttgcaca gaatagtaca   4500
atgaactcat atactgttca tctggattca ccaattgtta gtagcctttc gcttcatagg   4560
tttcacatct cttccctccg tctcttaccg tgctgcccac acactcacac acacacactc   4620
acacacacat acggatatat gtttactgtt attaatgctg aattgtctcg ataaagtttc   4680
agggattatg gtcctttacc ctatgtactt gagggtgtgt atatcgtcag aacaaagaga   4740
aagtcatttc ttggatcatc actgcacaaa gataaaaatc aggaaattta acaatgagaa   4800
aatggagtca tttaatcaca gagtgcatac tcaaattttc ccagttcccc agaaaatttc   4860
ttttttcctt ttttttttct ttgttgagac ggagtctccc tctgtgggcc aggttggagg   4920
gcagtagtgc gatctcggct cactgcaacc tacacctccc aggttctagg gattctcatg   4980
cctcagcctc ccgtgtagct gggactacag gcgccggcca ctgcggtctt gaacttctgg   5040
cctcacctgc tctgcccacc ttggcatccc aaaatgtttg gattgcaggc gtgagacccc   5100
acgcccggcc cagataattt tattgatagg atttcttttt ctgatccaga gtccagttga   5160
gaatcacacc ttgcatgtgc ctttcaggtg tttttagttt cctttaacct gtaatgtttc   5220
cttaattttt cttgtcattc acgatacgga catttttgga gaggatagac cagttggttt   5280
gcagaatatt ctgtagtttg ggctttttca tgtatttttt aaaagagttt tctcactcag   5340
cgtttattgg tggctactca tgccatgtaa gagtctaagc gctaggagtg taagtgctgt   5400
gagagacggg atttgagcct tgagtcattt aatacgagaa ggacaatcag aagtagaata   5460
agagagaagt gcaaaggagg cagcaaagtt gtctgagggc agtcttcgga aaggaggagg   5520
gtaatatttc gaacaccttg ttttcctgtt ttctgctaac ggactcctga aataatgttc   5580
ctgggattct tatcaacaca tttattatta cgttagctaa agctctttat ataataatac   5640
cgagagcatg aatatcattt tcttattcat attttatgtt ttactgctta aattgatacg   5700
tattttttat ttttaagggc cgaagcctga agctcatagc caggaacagg gtcacccaca   5760
gactgggtgt gagtgtgaag atggtcctga tgggcaggag atggacccgc caaatccaga   5820
ggaggtgaaa acgcctgaag aaggtaggca atccattagg catgcacatt gtagggtgtc   5880
tgtttccaca gtatcatatt gtaactctta ctatgttttt gagacggagt ctcgctctga   5940
agaccaggct ggagtgcagt ggtgccatct cggctcactg gaaattctgt ctccagggtt   6000
caagtgattc tcctgcctga gcctctggcg gagccgggct tacaggcatg ctccgccgcg   6060
cccagctaat tgttgtattt ttagtagaga cagggtttcg ttatgttgca caggttgttc   6120
ccgaactcct gacctcaggt gatccacctg cctcgaccat tgaaattgcc gggattacag   6180
gcagagccac cgtgcccgac ccagcattat atttttaata acagagaggt aacaatactg   6240
cgtctttagt aacagagttc ttatataaag gttatttgaa acgtagttca ggccccagca   6300
cccggctgat agactgtcag atagggaaac aaagtgagtc aaagctatgt tgaattaaaa   6360
gttttgagta taaatcctta aaccagtagc tcacaatttt cagatgcttt tgtaaaggtc   6420
tgcttttaat caatacataa cacgtttgta acacccatca cttggtgtga aaaatgctga   6480
agcactcatg cgggttctaa taccagctct tacagccttg gcgagattct gagtgagtcc   6540
tttcccttct aaacctatct ttggttctta tgaaaatagt gagtttaagt cagagacttt   6600
aaaaccattt tgcattccgt ttctttcata ctctgatcct gttgcataga atgcgtggga   6660
cacagagatc atctcttcgc atggtttgtt aatcacaaat catgaaaccc tggcccgagt   6720
catctgaaaa tctctgaatt gagatttcat tgtcagtaag acagtgagcg ggccctctgc   6780
ttcatcctag tttttccgtg tggagagctg aatacgtagt ataagatctt gtgaaattgt   6840
gaattctccc tcttcttggt ttgtttgttt gtttgcgaca gagtctcagt gtgtcaccca   6900
ggctggagtg cagtgatgca atttcagctc actgcaactt ctggctccca gctaaagccg   6960
tcctcccacc tcagcctccc gagtggctgg aactacatgc acaagccacc gtgcctgact   7020
acattttttt gttttcattt ttgtagagat gaggtctcac tgtgttgccc aggcagggtt   7080
tctctggctt ttaatgaaca attgcttctt ttttttcctt ttatttattt attatacttt   7140
aagttttagg gtacatgtga cgttgtgcag gttagttaca tacgtataca tgtgccatgc   7200
tgtgcgctgc acccactatc tcatcatcta gcattaggta catctcccag tgctatccct   7260
cccccctccc cccacccgac aacagtcccc agggtgtgat attccccttc ctctgtccat   7320
gtgatctcat tgttcagttc ccacctatga gtgagaatat gcggtgtttg gttttttgtt   7380
cttgcgatag tttactgaga atgatgattt cnagtttcat ccatgtccct acaaaggaca   7440
tgaactcttc attttttagg gctgcatagt attccatagt gtatatgtgc cacattttct   7500
taatccagtc tatcgttgtt ggacatttgg gttggttcca agtctttgct atcgtgaata   7560
atgccgcaat aaacatacgt gtgcacgtgt ctttatagca gcatgattta tagtcctttg   7620
ggtatatacc cagtaatggg atggctgggt caaatggtac aattgcttct taaatctttc   7680
cccacggaaa ccttgagtga ctgaaataaa tatcaaatgg cgagagaccg tttagttcgt   7740
atcatctgtg gcatgtaggt cagtgatgct cagcatgggt gtgagtaaga tgcctgtgct   7800
atgcatgctc cctgccccac tgtcagtctt catgagccac tatttctaat aagactgtag   7860
acacacatac gatataatca tctctaatca tatcaaatgt tacatgtaag tttcagcttt   7920
agagacatga attgataaga tttaaagttg aaagaccatg actctagtac ttcctgagta   7980
atcaactgaa gtatgcttta cacatgtgtt ttccaaattg ctgactgtta attgtaagtg   8040
cttgtgactt gaaaggaagc acatgatgtt cagggaggaa attcctttta aattctgcag   8100
gtctacgctc aaagtttatg cagaggttca attgcgtgta agacacggga tcacccatag   8160
ggttctgttt ttagtccatt taataaaacc caaactgtag tgtgctttgt atgcctttag   8220
ggtcatctga ataatctgtt gctaagtcat gttcccaatc gttgtgtttc tgttacaggt   8280
gaaaagcaat cacagtgtta aaagaaggca cgttgaaatg atgcaggctg ctcctatgtt   8340
ggaaatttgt tcattaaaat tctcccaata aagctttaca gccttctgca aagaagtctt   8400
gcgcatcttt tgtgaagttt atttctagct ttttgatgct gtgaaatatg tatcattctt   8460
tgaaatcgtg tattgtaact ctctgagctg gtatgtagag acatcgttct tttttttttt   8520
ctttctttct ttgtcctctt ttgagacgga gtcttgctct gtcgcccagg ctggagtgca   8580
gtggcgcgat ctctgctcac tgcaaccccg cctcccggat tcaagcaatt gtctgcctca   8640
gcctcccgag tagctgggat tataggcacc caccagcacg ccctggctaa gttttgtgtt   8700
tttactagag atggtttcgc atcttggccg gggtgctctt gaactcctga cctcgtgatt   8760
cacctgcctt ggcctcccaa agtgctggga ttacaggcat gcacgcctcc gcgcccggtg   8820
gagacataat tcttacatat tggttttcta tccagcggcc ttgtgaaata tgcttgtgaa   8880
ttctaaagtt tacttctagg tcgttttcag tcttcaatat acagaaacat atcatcctgg   8940
aataagagca gttttgtttc cgccattttt ttttgttttt ccttttgtac tttttttgta   9000
gagacggggt tttgccatgt ttcccgggct gttgttgnnn ttttgagtgc aagtgatgca   9060
cccacgtcac ctcccacagt gctgggatta ctggcgtggg ccaggggcca cccgtggcgg   9120
gccccgtcgt tgccattgta aagagtttta tttccttttc tgattttatg gcattgcgca   9180
gacccacccg ttacaatggt gacagtggac atccttgtct tatccctgat gagaaaccga   9240
aaaatttcaa catttcgcca tcctattcac tctccttttt ttgtagacgg actttatcag   9300
agtgagtcat tgcattctgt tccaaatttg ctgagagtat tcatttgaat atatgttgat   9360
tttcatcaaa cagtgcatct atttcgatta ccacagcgtt ttttcccatt catgggttaa   9420
tatagtgaat tcgattgata aatttgtacg tttttaggtt cgattattaa aacttgagac   9480
agcgtctcac tctgtcaccg aggctggagt gcggtggtgt tatcagagct c            9531
 
           
             4 
             18 
             DNA 
             Homo sapiens 
             
 
           
            4
gaccaagacg  ctacgtag                                                  18
 
           
             5 
             18 
             DNA 
             Homo sapiens 
             
 
           
            5
ccatcaggac  catcttca                                                  18
 
           
             6 
             18 
             DNA 
             Homo sapiens 
             
 
           
            6
gaccaaggcg  ctatgtac                                                  18
 
           
             7 
             117 
             PRT 
             Homo sapiens 
             
 
           
            7
Met Ser Trp Arg Gly Arg Ser Thr Tyr Tyr Trp Pro Arg Pro Arg Arg
 1               5                  10                  15
Tyr Val Gln Pro Gly Pro Met Arg Pro Glu Gln Phe Ser Asp Glu Val
            20                  25                   30
Pro Glu Met Ile Glu Pro Ala Thr Pro Glu Glu Gly Glu Pro Ala Thr
        35                  40                   45
Gln Arg Gln Asp Pro Ala Ala Ala Gln Glu Gly Glu Asp Glu Gly Ala
     50                  55                  60
Ser Ala Gly Gln Gly Pro His Pro Gln Thr Gly Lys Pro Glu Ala His
 65                  70                  75                  80
Ser Gln Glu Gln Gly Cys Glu Cys Glu Asp Gly Pro Asp Gly Gln Glu
                 85                  90                  95
Met Asp Pro Pro Asn Pro Glu Glu Val Lys Thr Pro Glu Glu Gly Glu
            100                 105                 110
Lys Gln Ser Gln Cys
        115
 
           
             8 
             116 
             PRT 
             Homo sapiens 
             
 
           
            8
Met Ser Trp Arg Gly Arg Ser Thr Tyr Arg Pro Arg Pro Arg Arg Tyr
 1               5                   10                  15
Val Glu Pro Pro Glu Met Ile Gly Pro Met Arg Pro Glu Gln Phe Ser
             20                  25                  30
Asp Glu Val Glu Pro Ala Thr Pro Glu Glu Gly Glu Pro Ala Thr Gln
         35                  40                  45
Arg Gln Asp Pro Ala Ala Ala Gln Glu Gln Glu Asp Glu Gly Ala Ser
     50                  55                  60
Ala Gly Gln Gly Pro Lys Pro Glu Ala Asp Ser Gln Glu Gln Gly His
65                   70                  75                  80
Pro Gln Thr Gly Cys Glu Cys Glu Asp Gly Pro Asp Gly Gln Glu Met
                 85                  90                  95
Asp Pro Pro Asn Pro Glu Glu Val Lys Thr Pro Glu Glu Lys Glu Lys
            100                 105                 110
Gln Ser Gln Cys
        115
 
           
             9 
             9 
             PRT 
             Homo sapiens 
             
 
           
            9
Glu Gly Glu Pro Ala Thr Gln Arg Gln
                 5
 
           
             10 
             9 
             PRT 
             Homo sapiens 
             
 
           
            10
Tyr Tyr Trp Pro Arg Pro Arg Arg Tyr
                5
 
           
             11 
             9 
             PRT 
             Homo sapiens 
             
 
           
            11
Arg Tyr Val Gln Pro Pro Glu Met Ile
                5
 
           
             12 
             9 
             PRT 
             Homo sapiens 
             
 
           
            12
Ile Gly Pro Met Arg Pro Glu Gln Phe
                5
 
           
             13 
             9 
             PRT 
             Homo sapiens 
             
 
           
            13
Trp Pro Arg Pro Arg Arg Tyr Val Gln
                5
 
           
             14 
             8 
             PRT 
             Homo sapiens 
             
 
           
            14
Tyr Pro Pro Met Ile Gly Pro Met
                5
 
           
             15 
             10 
             PRT 
             Homo sapiens 
             
 
           
            15
Met Ser Trp Arg Gly Arg Ser Asp Glu Val
                5                   10
 
           
             16 
             9 
             PRT 
             Homo sapiens 
             
 
           
            16
Arg Pro Glu Gln Phe Ser Asp Glu Val
                5
 
           
             17 
             9 
             PRT 
             Homo sapiens 
             
 
           
            17
Asp Glu Val Glu Pro Ala Thr Pro Glu
                5
 
           
             18 
             9 
             PRT 
             Homo sapiens 
             
 
           
            18
Gln Glu Gly Glu Asp Glu Gly Ala Ser
                5
 
           
             19 
             9 
             PRT 
             Homo sapiens 
             
 
           
            19
Glu Glu Gly Glu Lys Gln Ser Gln Cys
                5
 
           
             20 
             9 
             PRT 
             Homo sapiens 
             
 
           
            20
Met Asp Pro Pro Asn Gln Glu Glu Val
                5
 
           
             21 
             9 
             PRT 
             Homo sapiens 
             
 
           
            21
Tyr Val Glu Pro Pro Glu Met Ile Gly
                5
 
           
             22 
             9 
             PRT 
             Homo sapiens 
             
 
           
            22
Thr Tyr Arg Pro Arg Pro Arg Arg Tyr
                5
 
           
             23 
             9 
             PRT 
             Homo sapiens 
             
 
           
            23
Arg Tyr Val Glu Pro Pro Glu Met Ile
                5
 
           
             24 
             9 
             PRT 
             Homo sapiens 
             
 
           
            24
Arg Pro Arg Pro Arg Arg Tyr Val Glu
                5
 
           
             25 
             9 
             PRT 
             Homo sapiens 
             
 
           
            25
Glu Pro Pro Glu Met Ile Gly Pro Met
                5
 
           
             26 
             8 
             PRT 
             Homo sapiens 
             
 
           
            26
Tyr Arg Pro Arg Pro Arg Arg Tyr
                5
 
           
             27 
             9 
             PRT 
             Homo sapiens 
             
 
           
            27
Tyr Tyr Trp Pro Arg Pro Arg Arg Tyr
                5