Patent Publication Number: US-5527897-A

Title: Human ID genes

Description:
FIELD OF THE INVENTION 
     The present invention relates to human Id genes, and more particularly to two genes, Id-1H and Id-1H&#39; (both genes will hereinafter be called &#34;human Id genes&#34;), which code for human Id proteins, respectively, expression vectors with the human Id genes separately integrated therein, transformed cell strains with the expression vectors separately introduced therein, recombinant Id proteins obtained by cultivating the respective transformed cells, diagnostic probes composed of respective partial sequences of the base sequences of the human Id genes, or variation sequences thereof, and antisense oligonucleotides composed of respective base sequences complementary to the partial sequences of the base sequences of the human Id genes, or the variation sequences thereof. The antisense oligonucleotides can be used in antisense therapy. 
     BACKGROUND OF THE INVENTION 
     An Id protein was found as a myogenic regulatory factor suppressing the activity of a muscle-specific gene in mouse C3H10T1/2 fibroblasts [Cell, Vol. 61, 49-59 (1991)]. A subsequent research revealed that Id proteins are recognized in many kinds of cells during proliferation and decrease upon removal of sera and growth factors, and differentiation of cells [Proc. Natl. Acad. Sci. USA. Vol. 88, 1815-1819 (1991)]. 
     From the above-described facts, the presence of an Id protein in cells and the expression of an Id gene that codes for the Id protein can be positioned as an index for determining the state of cell proliferation, and the state of cell differentiation and aging. 
     Three kinds of genes, Id-1, Id-2 and HLH462 have presently been known as mouse Id genes. However, only a human Id-2 gene corresponding to the mouse Id-2 gene has heretofore been known as an Id gene present in human cells [Proc. Natl. Acad. Sci. USA, Vol. 89, 1512-1516 (1992)]. Therefore, several unknown human Id genes have been considered to exist. In view of further applications of Id genes and knowledge about the Id genes to fundamental experiments on the human and human cells, diagnoses and therapies, a problem to be early overcome has been to acquire unknown human Id genes. 
     OBJECTS AND SUMMARY OF THE INVENTION 
     It is an object of the present invention to provide a novel human Id gene. 
     Another object of the present invention is to provide an expression vector with the human Id gene integrated therein, a transformed cell strain with the expression vector introduced therein, a recombinant Id protein obtained by cultivating the transformed cells, a diagnostic probe composed of a partial sequence of the base sequence of the human Id gene, or a variation sequence thereof, and an antisense oligonucleotide composed of a base sequence complementary to the partial sequence of the base sequence of the human Id gene, or the variation thereof. 
     The present inventors have carried out an extensive investigation with a view toward solving the above-described problem. As a result, Id-1H and Id-1H&#39; have been successfully isolated as novel human Id genes corresponding to the mouse Id-1 gene. These two human Id genes are derived from the same genetic locus on a genome by alternate splicing. The isolated human Id gene has been integrated in an expression vector which has then been introduced into Escherichia coli cells, thereby successfully producing a recombinant Id protein in a great amount. The present inventors have further found that the expression of the Id-1H and Id-1H&#39; genes specifically varies according to the state of cell proliferation, in particular, the acquisition of cell aging and permanently proliferating ability, and revealed that these Id genes can be used in diagnostic probes and the like, thus leading to completion of the present invention. 
     According to the present invention, there are thus provided a human Id gene (Id-1H) represented by a base sequence of SEQ ID NO:1 in Sequence Table which will be described subsequently, and a human Id gene (Id-1H&#39;) represented by a base sequence of SEQ ID NO:3. These human Id genes include respective variants. 
     According to the present invention, there are also provided novel expression vectors with the human Id genes, or the variants thereof separately integrated therein, and novel transformed cell strains with the expression vectors separately introduced therein. 
     According to the present invention, there are further provided recombinant Id proteins obtained by separately cultivating the transformed cell strains. 
     Diagnostic probes can be obtained by chemically synthesizing a partial sequence of the base sequence of each of the human Id genes, or a variation sequence thereof. When an antisense oligonucleotide composed of a base sequence complementary to the partial sequence of the base sequence of the human Id gene, or the variation sequence thereof is synthesized, it can be used in antisense therapy and the like. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 illustrates a base sequence of an oligonucleotide used as a probe upon screening two human Id genes (Id-1H and Id-1H&#39;) from a human cDNA library by the hybridization method; 
     FIG. 2 illustrates a base sequence of another oligonucleotide used as a probe upon screening two human Id genes (Id-1H and Id-1H&#39;) from the human cDNA library by the hybridization method; 
     FIG. 3 is a graph illustrating variations in quantity of the transcription product by the Id-1H gene in juvenile cells according to the state of proliferation; 
     FIG. 4 is a graph illustrating variations in quantity of the transcription product by the Id-1H gene in senile cells according to the state of proliferation; 
     FIG. 5 is a graph illustrating variations in quantity of the transcription product by the Id-1H&#39; gene in juvenile cells according to the state of proliferation; and 
     FIG. 6 is a graph illustrating variations in quantity of the transcription product by the Id-1H&#39; gene in senile cells according to the state of proliferation. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention will hereinafter be described in detail. 
     (1) Isolation of Human Id Gene 
     The human Id genes (Id-1H and Id-1H&#39;) according to the present invention are cDNAs derived from human TIG-1 fibroblasts. Each of these cDNAs can be prepared in the following manner. 
     An oligonucleotide having a base sequence complementary to a base sequence which is considered to be well-conserved among DNA sequences of three kinds of the known mouse Id genes (Id-1, Id-2 and HLH462) and be indispensable to proteins from the viewpoint of the corelation between their structures and functions is first synthesized. The thus-synthesized oligonucleotide is then used as a probe to screen a clone containing a cDNA complementary to the probe from a cDNA library of the human TIG-1 fibroblasts by the hybridization method, thereby isolating a human Id gene. 
     Base sequences of two synthetic oligonucleotides (HLH-1 and HLH-2) used as the probes are illustrated in FIGS. 1 and 2, respectively. These two base sequences are base sequences (each 38-mer) corresponding to two regions of amino acid sequence (corresponding to two α-helix portions), which are almost completely conserbed by the three mouse Id genes. Since the synthetic oligonucleotides are synthesized by means of an oligonucleotide synthesizer on the basis of the amino acid sequences, they are obtained as mixtures of those containing different base sequences due to degeneracy, as shown in FIGS. 1 and 2. 
     With respect to two kinds of the human Id genes thus obtained, all base sequences are determined by the dideoxynucleotide chain-termination method or Maxam-Gilbert method, and their portions required for the expression of the human Id genes are then determined. The base sequence thus determined are shown as Sequence No. 1 (Id-1H) and Sequence No. 2 (Id-1H&#39;) in Sequence Table which will be described below. The human Id genes according to the present invention include these two genes, Id-1H and Id-1H&#39; and variants thereof 
     (2) Production of Human Id Gene-integrated Expression Vector, Transformed Cell with the Expression Vector Introduced Therein, and Human Id Protein 
     Each of the human Id genes (cDNAs) isolated in the above-described manner is integrated in a vector by a method known per se in the art. Vectors used may be those lacking for a DNA portion other than a portion required for proliferation in addition to extracts from naturally occurred products. For example, a pET3 vector may preferably be used for mass production of human Id proteins. As a method for integrating the cDNA in the vector DNA, there may be used any known method, for example, treatment of the DNA with an restriction enzyme and reconstitution with a ligase. 
     The human Id gene-integrated plasmid vector thus obtained is introduced into host microorganisms by a method known per se in the art to obtain a transformed cell strain (transformant). As the host microorganisms to be introduced, it is preferable to use microorganisms (Escherichia coli) belonging to Escherichia and typified by an Escherichia coli K12 strain. If the pET3 vector is used as an expression vector for producing a human Id protein, it is particularly preferable to use BL21(DE3)Lys S as microorganisms to be introduced because impurities derived from Escherichia coli remain little in the course of purification, and time and labor can hence be saved. 
     These transformed cell strains are novel microorganisms obtained by transformation in which a plasmid vector obtained by integrating the human Id gene (cDNA, Id-1H or Id-1H&#39;) in a pUC118 plasmid is introduced into the Escherichia coli K12 strain and include two strains called &#34;E. coli pId-1H&#34; (NIBHT Deposition FERM BP-4460 deposited in National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-3, Higashi 1 chome Tsukuba-shi Ibaraki-ken 305, Japan: Escherichia coli K12 strain) obtained by using Id-1H as cDNA and &#34;E. coli pId-1H&#39;&#34; (NIBHT Deposition FERM BP-4461: Escherichia coli K12 strain) obtained by using Id-1H&#39; as cDNA. 
     Similarly, a transformed cell strain can be obtained by introducing an expression vector, which has been obtained by integrating the human Id gene (cDNA, Id-1H or Id-1H&#39;) in an expression vector, for example, a pET3 vector or the like, into Escherichia coli, for example, BL21(DE3)Lys S. This strain can be cultivated, thereby obtaining a recombinant Id protein from the resultant culture. 
     (3) Diagnostic Probe and Antisense Oligonucleotide 
     The Id proteins are recognized to not only have properties as myogenic regulatory factors which serve to suppress the activity of muscle-specific genes, but also exist widely in actively proliferating cells. The quantity of the Id proteins decreases when proliferation is terminated by removing sera and growth factors or inducing cell differentiation. 
     Further, the present inventors have newly found that the human Id proteins specifically varies according to the acquisition of cell aging and permanently proliferating ability. Therefore, it is thought that information for determining the state of proliferation, and the state of differentiation and aging in tissues and cells can be obtained by knowing the quantity of Id genes expressed in cells and the presence of mutation of Id loci in genomes, and that a partial sequence of a human Id gene sequence, or a variation sequence thereof can be used as a diagnostic probe. 
     The Id proteins are also transcriptional regulatory factors existing in nuclei, and there is hence a possibility that they may regulate the expression of a great number of genes participating in cell proliferation in view of their mechanism. In that case, it is thought that the expression of a wide range of genes participating in cell proliferation can be affected by controlling the expression of the Id genes. If a means for effectively inhibiting the synthesis of Id proteins can be found by an antisense technique or the like, a therapeutic means can be provided for diseases, in which proliferation aberration acquires an important significance, such as cancer and progeria. For example, in an experiment making use of cultured cells, cell proliferation can be controlled negatively by inhibiting the expression of an Id gene with an antisense oligonucleotide. 
     ADVANTAGES OF THE INVENTION 
     According to the present invention, there are provided novel human Id genes, expression vectors with the human Id genes separately integrated therein, transformed cell strains with the expression vectors separately introduced therein, and the like. The human Id genes according to the present invention are useful in a field of genetic engineering, fundamental experiments on human cells, diagnoses, therapies and the like. For example, they can be expected to apply to diagnostic probes composed of respective partial sequences of the base sequences of the human Id genes, or variation sequences thereof, and antisense therapy using antisense oligonucleotides composed of respective base sequences complementary to the partial sequences of the base sequences of the human Id genes, or the variation sequences thereof. 
     EMBODIMENTS OF THE INVENTION 
     The present invention will hereinafter be described more specifically by the following examples. 
     EXAMPLE 1 
     (1) Selection and Chemical Synthesis of Base Sequence Used as Probe 
     In order to isolate human Id genes existing in human cells, amino acid sequences coded by three mouse Id genes (Id-1, Id-2 and HLH462) were investigated to find two regions of amino acid sequence (corresponding to two α-helix portions) which were almost completely conserved by the three mouse Id genes. Two base sequences (each 38-mer) corresponding to the amino acid sequence regions were determined (see FIGS. 1 and 2). Two oligonucleotides (HLH-1 and HLH-2) respectively having the base sequences determined in the above-described manner were synthesized by means of an oligonucleotide synthesizer (380B) manufactured by Applied Biosystems Company. 
     (2) Construction of Human cDNA Library 
     A cDNA library was constructed using normal human TIG-1 fibroblasts. The Okayama-Berg method was used in the construction of the cDNA library to obtain a cDNA library composed of 1×10 6  colonies. 
     (3) Cloning of Human Id Gene cDNA 
     The human cDNA library constructed by the above-described process was screened using, as a probe, a mixture of two kinds of the 38-mer oligonucleotides synthesized in the step (1). The 5&#39; terminal of each of the oligonucleotides was labeled with [γ- 32  P]ATP to hybridize it to a human cDNA fixed to a nylon filter for 16 hours at 65° C. As a reaction solution, was used a mixed solution composed of 5×Denhardt&#39;s solution, 5×SSC and 0.5% SDS. 
     After the hybridization, the filter was washed each twice with (1) a wash of 2×SSC for 10 minutes at room temperature, (2) a wash composed of 2×SSC and 0.1% SDS for 10 minutes at 65° C. and then (3) a wash composed of 0.1×SSC and 0.1% SDS for 5 minutes at room temperature. After drying, exposure was conducted with an X-ray film (Kodak XAR-5) for 7 days at -80° C. Assay was conducted on 10 6  colonies, and found to obtain ten positive clones. 
     (4) Determination of Human Id Gene Base Sequence 
     Three clones (Clone Nos. 1-3) among the ten clones obtained in the step (3), which did not overlap with each other, were recloned in a pUC118 plasmid. Thereafter, the base sequences of these three clones were determined by means of a Sequenare Version 2 (manufactured by USB Co.) in accordance with the dideoxynucleotide chain-termination method. As a result, it was found that the base sequence of Clone No. 3 is the same as that of the human Id-2 gene already known, but Clone No. 1 and Clone No. 2 are new genes. The base sequence of Clone No. 1 had a homology of 85% to that of the mouse Id gene (Id-1). Therefore, Clone No. 1 was designated as &#34;Id-1H&#34; (H means human). Since Clone No. 2 is considered to be a product created from the same genetic locus as Clone No. 1 by alternate splicing, it was designated as &#34;Id-1H&#39;&#34;. The determined base sequences of the Id-1H and Id-1H&#39; genes are shown in Sequence Table which will be described subsequently. 
     EXAMPLE 2 
     Preparation of New Microorganisms 
     With respect to Id-1H, a pCD2 plasmid containing the Id-1H gene derived from the cDNA library constructed in Example 1 was cleaved by an restriction enzyme BamH I to take out an Id-1H cDNA, which was then integrated into a BamH I site of a pCU118 plasmid. 
     With respect to Id-1H&#39;, a pCD2 plasmid containing the Id-1H&#39; gene derived from the cDNA library constructed in Example 1 was cleaved by restriction enzymes BamH I and Kpn I to take out Id-1H&#39; cDNAs, which were then integrated into BamH I and Kpn I sites of a pCU118 plasmid. Thus, two kinds of new microorganisms were obtained. One is E. coli pId-1H (NIBHT Deposition FERM BP-4460) obtained by using Id-1H and the other is E. coli pId-1H&#39; (NIBHT Deposition FERM BP-4461) obtained by using Id-1H&#39;. Similarly, expression vectors obtained by separately integrating the two human Id genes into an expression vector pET3 were introduced into BL21(DE3)Lys S to obtain respective transformed cell strains. 
     EXAMPLE 3 
     Variations in Quantity of the Transcription Product by Id Gene in Cells According to the State of Proliferation 
     Variation in quantity of the transcription products (mRNA) by the Id-1H and Id-1H&#39; genes according to the state of proliferation were determined in accordance with the Northern blot hybridization method (see FIGS. 3 through 6). As illustrated in FIGS. 3 and 4, the quantities of mRNA produced by Id-1H (FIG. 3) and Id-1H&#39; (FIG. 4) as to juvenile cells show specific variation patterns according to the lapse of time. On the contrary, as illustrated in FIGS. 5 and 6, the quantities of mRNA produced by Id-1H (FIG. 5) and Id-1H&#39; (FIG. 6) as to senile cells remain substantially zero corresponding to the fact that the cells come not to respond to proliferative stimulation. These experimental results suggest that the Id-1H and Id-1H&#39; genes bear a certain action in the course of proliferation control and aging. 
     Therefore, partial sequences of the base sequences of the human Id genes (Id-1H and Id-1H&#39;), or variation sequences thereof are useful as diagnostic probes. If antisense oligonucleotides composed separately of base sequences complementary to the partial sequences of the base sequences of the human Id genes, or the variation sequences thereof are synthesized, they are useful in antisense therapy. 
     
                                           SEQUENCE TABLE                          
__________________________________________________________________________
SEQ ID NO: 1                                                              
Length of sequence: 509                                                   
Type of sequence: nucleic acid                                            
Number of chains: double strand                                           
Topology: linear                                                          
Kind of sequence: cDNA to mRNA                                            
Origin:                                                                   
   Name of organism: Home sapiens                                         
   Kind of cell: TIG-1                                                    
Feature of sequence:                                                      
   Symbol indicating feature: CDS                                         
   Existing position: 22..486                                             
   Characterizing method: P                                               
Sequence:                                                                 
TTCAGCCAGT CGCCAAGAAT C                                                   
                  ATG                                                     
                     AAA                                                  
                        GTC                                               
                           GCC                                            
                              AGT                                         
                                 GGC                                      
                                    AGC                                   
                                       ACC                                
                                          GCC                             
                                             48                           
                  Met                                                     
                     Lys                                                  
                        Val                                               
                           Ala                                            
                              Ser                                         
                                 Gly                                      
                                    Ser                                   
                                       Thr                                
                                          Ala                             
                  1           5                                           
ACC                                                                       
   GCC                                                                    
      GCC                                                                 
         GCG                                                              
            GGC                                                           
               CCC                                                        
                  ACG                                                     
                     TGC                                                  
                        GCG                                               
                           CTG                                            
                              AAG                                         
                                 GCC                                      
                                    GGC                                   
                                       AAG                                
                                          ACA                             
                                             GCG 96                       
Thr                                                                       
   Ala                                                                    
      Ala                                                                 
         Ala                                                              
            Gly                                                           
               Pro                                                        
                  Thr                                                     
                     Cys                                                  
                        Ala                                               
                           Leu                                            
                              Lys                                         
                                 Ala                                      
                                    Gly                                   
                                       Lys                                
                                          Thr                             
                                             Ala                          
10             15             20             25                           
AGC                                                                       
   GGT                                                                    
      GCG                                                                 
         GGC                                                              
            GAG                                                           
               GTG                                                        
                  GTG                                                     
                     CGC                                                  
                        TGT                                               
                           CTG                                            
                              TCT                                         
                                 GAG                                      
                                    CAG                                   
                                       AGC                                
                                          GTG                             
                                             GCC 144                      
Ser                                                                       
   Gly                                                                    
      Ala                                                                 
         Gly                                                              
            Glu                                                           
               Val                                                        
                  Val                                                     
                     Arg                                                  
                        Cys                                               
                           Leu                                            
                              Ser                                         
                                 Glu                                      
                                    Gln                                   
                                       Ser                                
                                          Val                             
                                             Ala                          
            30             35             40                              
ATC                                                                       
   TCG                                                                    
      CGC                                                                 
         TGC                                                              
            CGG                                                           
               GGC                                                        
                  GCC                                                     
                     GGG                                                  
                        GCG                                               
                           CGC                                            
                              CTG                                         
                                 CCT                                      
                                    GCC                                   
                                       CTG                                
                                          CTG                             
                                             GAC 192                      
Ile                                                                       
   Ser                                                                    
      Arg                                                                 
         Cys                                                              
            Arg                                                           
               Gly                                                        
                  Ala                                                     
                     Gly                                                  
                        Ala                                               
                           Arg                                            
                              Leu                                         
                                 Pro                                      
                                    Ala                                   
                                       Leu                                
                                          Leu                             
                                             Asp                          
         45             50             55                                 
GAG                                                                       
   CAG                                                                    
      CAG                                                                 
         GTA                                                              
            AAC                                                           
               GTG                                                        
                  CTG                                                     
                     CTC                                                  
                        TAC                                               
                           GAC                                            
                              ATG                                         
                                 AAC                                      
                                    GGC                                   
                                       TGT                                
                                          TAC                             
                                             TCA 240                      
Glu                                                                       
   Gln                                                                    
      Gln                                                                 
         Val                                                              
            Asn                                                           
               Val                                                        
                  Leu                                                     
                     Leu                                                  
                        Tyr                                               
                           Asp                                            
                              Met                                         
                                 Asn                                      
                                    Gly                                   
                                       Cys                                
                                          Tyr                             
                                             Ser                          
      60             65             70                                    
CGC                                                                       
   CTC                                                                    
      AAG                                                                 
         GAG                                                              
            CTG                                                           
               GTG                                                        
                  CCC                                                     
                     ACC                                                  
                        CTG                                               
                           CCC                                            
                              CAG                                         
                                 AAC                                      
                                    CGC                                   
                                       AAG                                
                                          GTG                             
                                             AGC 288                      
Arg                                                                       
   Leu                                                                    
      Lys                                                                 
         Glu                                                              
            Leu                                                           
               Val                                                        
                  Pro                                                     
                     Thr                                                  
                        Leu                                               
                           Pro                                            
                              Gln                                         
                                 Asn                                      
                                    Arg                                   
                                       Lys                                
                                          Val                             
                                             Ser                          
   75             80             85                                       
AAG                                                                       
   GTG                                                                    
      GAG                                                                 
         ATT                                                              
            CTC                                                           
               CAG                                                        
                  CAC                                                     
                     GTC                                                  
                        ATC                                               
                           GAC                                            
                              TAC                                         
                                 ATC                                      
                                    AGG                                   
                                       GAC                                
                                          CTT                             
                                             CAG 336                      
Lys                                                                       
   Val                                                                    
      Glu                                                                 
         Ile                                                              
            Leu                                                           
               Gln                                                        
                  His                                                     
                     Val                                                  
                        Ile                                               
                           Asp                                            
                              Tyr                                         
                                 Ile                                      
                                    Arg                                   
                                       Asp                                
                                          Leu                             
                                             Gln                          
90             95             100            105                          
TTG                                                                       
   GAG                                                                    
      CTG                                                                 
         AAC                                                              
            TCG                                                           
               GAA                                                        
                  TCC                                                     
                     GAA                                                  
                        GTT                                               
                           GGA                                            
                              ACC                                         
                                 CCC                                      
                                    GGG                                   
                                       GGC                                
                                          CGA                             
                                             GGG 384                      
Leu                                                                       
   Glu                                                                    
      Leu                                                                 
         Asn                                                              
            Ser                                                           
               Glu                                                        
                  Ser                                                     
                     Glu                                                  
                        Val                                               
                           Gly                                            
                              Thr                                         
                                 Pro                                      
                                    Gly                                   
                                       Gly                                
                                          Arg                             
                                             Gly                          
            110            115            120                             
CTG                                                                       
   CCG                                                                    
      GTC                                                                 
         CGG                                                              
            GCT                                                           
               CCG                                                        
                  CTC                                                     
                     AGC                                                  
                        ACC                                               
                           CTC                                            
                              AAC                                         
                                 GGC                                      
                                    GAG                                   
                                       ATC                                
                                          AGC                             
                                             GCC 432                      
Leu                                                                       
   Pro                                                                    
      Val                                                                 
         Arg                                                              
            Ala                                                           
               Pro                                                        
                  Leu                                                     
                     Ser                                                  
                        Thr                                               
                           Leu                                            
                              Asn                                         
                                 Gly                                      
                                    Glu                                   
                                       Ile                                
                                          Ser                             
                                             Ala                          
         125            130            135                                
CTG                                                                       
   ACG                                                                    
      GCC                                                                 
         GAG                                                              
            GCG                                                           
               GCA                                                        
                  TGC                                                     
                     GTC                                                  
                        CCT                                               
                           GCG                                            
                              GAC                                         
                                 GAT                                      
                                    CGC                                   
                                       ATC                                
                                          TTG                             
                                             TGT 480                      
Leu                                                                       
   Thr                                                                    
      Ala                                                                 
         Glu                                                              
            Ala                                                           
               Ala                                                        
                  Cys                                                     
                     Val                                                  
                        Pro                                               
                           Ala                                            
                              Asp                                         
                                 Asp                                      
                                    Arg                                   
                                       Ile                                
                                          Leu                             
                                             Cys                          
      140            145            150                                   
CGC                                                                       
   TGAAGGCCTT                                                             
            CCCCAGGGAC                                                    
                     CGGCGG                                               
                           509                                            
Arg                                                                       
SEQ ID NO: 3                                                              
Length of sequence: 502                                                   
Type of sequence: nucleic acid                                            
Number of chains: double strand                                           
Topology: linear                                                          
Kind of sequence: cDNA to mRNA                                            
Origin:                                                                   
   Name of organism: Home sapiens                                         
   Kind of cell: TIG-1                                                    
Feature of sequence:                                                      
   Symbol indicating feature: CDS                                         
   Existing position: 22..486                                             
   Characterizing method: P                                               
Sequence:                                                                 
TTCAGCCAGT CGCCAAGAAT C                                                   
                  ATG                                                     
                     AAA                                                  
                        GTC                                               
                           GCC                                            
                              AGT                                         
                                 GGC                                      
                                    AGC                                   
                                       ACC                                
                                          GCC                             
                                             48                           
                  Met                                                     
                     Lys                                                  
                        Val                                               
                           Ala                                            
                              Ser                                         
                                 Gly                                      
                                    Ser                                   
                                       Thr                                
                                          Ala                             
                  1           5                                           
ACC                                                                       
   GCC                                                                    
      GCC                                                                 
         GCG                                                              
            GGC                                                           
               CCC                                                        
                  ACG                                                     
                     TGC                                                  
                        GCG                                               
                           CTG                                            
                              AAG                                         
                                 GCC                                      
                                    GGC                                   
                                       AAG                                
                                          ACA                             
                                             GCG 96                       
Thr                                                                       
   Ala                                                                    
      Ala                                                                 
         Ala                                                              
            Gly                                                           
               Pro                                                        
                  Thr                                                     
                     Cys                                                  
                        Ala                                               
                           Leu                                            
                              Lys                                         
                                 Ala                                      
                                    Gly                                   
                                       Lys                                
                                          Thr                             
                                             Ala                          
10             15             20             25                           
AGC                                                                       
   GGT                                                                    
      GCG                                                                 
         GGC                                                              
            GAG                                                           
               GTG                                                        
                  GTG                                                     
                     CGC                                                  
                        TGT                                               
                           CTG                                            
                              TCT                                         
                                 GAG                                      
                                    CAG                                   
                                       AGC                                
                                          GTG                             
                                             GCC 144                      
Ser                                                                       
   Gly                                                                    
      Ala                                                                 
         Gly                                                              
            Glu                                                           
               Val                                                        
                  Val                                                     
                     Arg                                                  
                        Cys                                               
                           Leu                                            
                              Ser                                         
                                 Glu                                      
                                    Gln                                   
                                       Ser                                
                                          Val                             
                                             Ala                          
            30             35             40                              
ATC                                                                       
   TCG                                                                    
      CGC                                                                 
         TGC                                                              
            CGG                                                           
               GGC                                                        
                  GCC                                                     
                     GGG                                                  
                        GCG                                               
                           CGC                                            
                              CTG                                         
                                 CCT                                      
                                    GCC                                   
                                       CTG                                
                                          CTG                             
                                             GAC 192                      
Ile                                                                       
   Ser                                                                    
      Arg                                                                 
         Cys                                                              
            Arg                                                           
               Gly                                                        
                  Ala                                                     
                     Gly                                                  
                        Ala                                               
                           Arg                                            
                              Leu                                         
                                 Pro                                      
                                    Ala                                   
                                       Leu                                
                                          Leu                             
                                             Asp                          
         45             50             55                                 
GAG                                                                       
   CAG                                                                    
      CAG                                                                 
         GTA                                                              
            AAC                                                           
               GTG                                                        
                  CTG                                                     
                     CTC                                                  
                        TAC                                               
                           GAC                                            
                              ATG                                         
                                 AAC                                      
                                    GGC                                   
                                       TGT                                
                                          TAC                             
                                             TCA 240                      
Glu                                                                       
   Gln                                                                    
      Gln                                                                 
         Val                                                              
            Asn                                                           
               Val                                                        
                  Leu                                                     
                     Leu                                                  
                        Tyr                                               
                           Asp                                            
                              Met                                         
                                 Asn                                      
                                    Gly                                   
                                       Cys                                
                                          Tyr                             
                                             Ser                          
      60             65             70                                    
CGC                                                                       
   CTC                                                                    
      AAG                                                                 
         GAG                                                              
            CTG                                                           
               GTG                                                        
                  CCC                                                     
                     ACC                                                  
                        CTG                                               
                           CCC                                            
                              CAG                                         
                                 AAC                                      
                                    CGC                                   
                                       AAG                                
                                          GTG                             
                                             AGC 288                      
Arg                                                                       
   Leu                                                                    
      Lys                                                                 
         Glu                                                              
            Leu                                                           
               Val                                                        
                  Pro                                                     
                     Thr                                                  
                        Leu                                               
                           Pro                                            
                              Gln                                         
                                 Asn                                      
                                    Arg                                   
                                       Lys                                
                                          Val                             
                                             Ser                          
   75             80             85                                       
AAG                                                                       
   GTG                                                                    
      GAG                                                                 
         ATT                                                              
            CTC                                                           
               CAG                                                        
                  CAC                                                     
                     GTC                                                  
                        ATC                                               
                           GAC                                            
                              TAC                                         
                                 ATC                                      
                                    AGG                                   
                                       GAC                                
                                          CTT                             
                                             CAG 336                      
Lys                                                                       
   Val                                                                    
      Glu                                                                 
         Ile                                                              
            Leu                                                           
               Gln                                                        
                  His                                                     
                     Val                                                  
                        Ile                                               
                           Asp                                            
                              Tyr                                         
                                 Ile                                      
                                    Arg                                   
                                       Asp                                
                                          Leu                             
                                             Gln                          
90             95             100            105                          
TTG                                                                       
   GAG                                                                    
      CTG                                                                 
         AAC                                                              
            TCG                                                           
               GAA                                                        
                  TCC                                                     
                     GAA                                                  
                        GTT                                               
                           GGA                                            
                              ACC                                         
                                 CCC                                      
                                    GGG                                   
                                       GGC                                
                                          CGA                             
                                             GGG 384                      
Leu                                                                       
   Glu                                                                    
      Leu                                                                 
         Asn                                                              
            Ser                                                           
               Glu                                                        
                  Ser                                                     
                     Glu                                                  
                        Val                                               
                           Gly                                            
                              Thr                                         
                                 Pro                                      
                                    Gly                                   
                                       Gly                                
                                          Arg                             
                                             Gly                          
            110            115            120                             
CTG                                                                       
   CCG                                                                    
      GTC                                                                 
         CGC                                                              
            GCT                                                           
               CCG                                                        
                  CTC                                                     
                     AGC                                                  
                        ACC                                               
                           CTC                                            
                              AAC                                         
                                 GGC                                      
                                    GAG                                   
                                       ATC                                
                                          AGT                             
                                             GCC 432                      
Leu                                                                       
   Pro                                                                    
      Val                                                                 
         Arg                                                              
            Ala                                                           
               Pro                                                        
                  Leu                                                     
                     Ser                                                  
                        Thr                                               
                           Leu                                            
                              Asn                                         
                                 Gly                                      
                                    Glu                                   
                                       Ile                                
                                          Ser                             
                                             Ala                          
         125            130            135                                
CTG                                                                       
   ACG                                                                    
      GCC                                                                 
         GAG                                                              
            GTG                                                           
               AGA                                                        
                  TCC                                                     
                     AGA                                                  
                        TCC                                               
                           GAC                                            
                              CAC                                         
                                 TAGATCATCC      480                      
Leu                                                                       
   Thr                                                                    
      Ala                                                                 
         Glu                                                              
            Val                                                           
               Arg                                                        
                  Ser                                                     
                     Arg                                                  
                        Ser                                               
                           Asp                                            
                              His                                         
      140            145                                                  
TTATACCGAC                                                                
         GGGGAAACGG                                                       
                   AGGCCAG                                                
                          502                                             
__________________________________________________________________________
 
    
     
         __________________________________________________________________________
SEQUENCE LISTING                                                          
(1) GENERAL INFORMATION:                                                  
(iii) NUMBER OF SEQUENCES: 8                                              
(2) INFORMATION FOR SEQ ID NO:1:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 509 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA to mRNA                                          
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(vi) ORIGINAL SOURCE:                                                     
(A) ORGANISM: Homo sapiens                                                
(ix) FEATURE:                                                             
(A) NAME/KEY: misc.sub.-- feature                                         
(B) LOCATION: 22..483                                                     
(D) OTHER INFORMATION: /note=&#34;CDS&#34;                                        
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                   
TTCAGCCAGTCGCCAAGAATCATGAAAGTCGCCAGTGGCAGCACCGCCACCGCCG CCGCG60           
GGCCCCACGTGCGCGCTGAAGGCCGGCAAGACAGCGAGCGGTGCGGGCGAGGTGGTGCGC120           
TGTCTGTCTGAGCAGAGCGTGGCCATCTCGCGCTGCCGGGGCGCCGGGGCGCGCCTGCCT180           
GCCCTGCTGGACGAGCAGCAGGTAAACGTG CTGCTCTACGACATGAACGGCTGTTACTCA240          
CGCCTCAAGGAGCTGGTGCCCACCCTGCCCCAGAACCGCAAGGTGAGCAAGGTGGAGATT300           
CTCCAGCACGTCATCGACTACATCAGGGACCTTCAGTTGCAGCTGAACTCGGAATCCGAA360           
GTTG GAACCCCCGGGGGCCGAGGGCTGCCGGTCCGGGCTCCGCTCAGCACCCTCAACGGC420          
GAGATCAGCGCCCTGACGGCCGAGGCGGCATGCGTCCCTGCGGACGATCGCATCTTGTGT480           
CGCTGAAGGCCTTCCCCAGGGACCGGCGG 509                                         
(2) INFORMATION FOR SEQ ID NO:2:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 154 amino acids                                               
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                   
MetLysValAlaSerGlySer ThrAlaThrAlaAlaAlaGlyProThr                         
151015                                                                    
CysAlaLeuLysAlaGlyLysThrAlaSerGlyAlaGlyGluValVal                          
20 2530                                                                   
ArgCysLeuSerGluGlnSerValAlaIleSerArgCysArgGlyAla                          
354045                                                                    
GlyAlaArgLeuProAlaL euLeuAspGluGlnGlnValAsnValLeu                         
505560                                                                    
LeuTyrAspMetAsnGlyCysTyrSerArgLeuLysGluLeuValPro                          
6570 7580                                                                 
ThrLeuProGlnAsnArgLysValSerLysValGluIleLeuGlnHis                          
859095                                                                    
ValIleAspTyrIle ArgAspLeuGlnLeuGluLeuAsnSerGluSer                         
100105110                                                                 
GluValGlyThrProGlyGlyArgGlyLeuProValArgAlaProLeu                          
115 120125                                                                
SerThrLeuAsnGlyGluIleSerAlaLeuThrAlaGluAlaAlaCys                          
130135140                                                                 
ValProAlaAspAspArgIl eLeuCysArg                                           
145150                                                                    
(2) INFORMATION FOR SEQ ID NO:3:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 502 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: cDNA to mRNA                                          
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
( ix) FEATURE:                                                            
(A) NAME/KEY: misc.sub.-- feature                                         
(B) LOCATION: 22..465                                                     
(D) OTHER INFORMATION: /note=&#34;CDS&#34;                                        
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                   
TTCAGCCAGTCGCCAAGAATCATGAAAGTCGCCAGTGGCAGCACCGCCACCGCCGCCGCG60            
GGCCCCACGTGCGCGCTGAAGGCCGGCAAGACAGC GAGCGGTGCGGGCGAGGTGGTGCGC120          
TGTCTGTCTGAGCAGAGCGTGGCCATCTCGCGCTGCCGGGGCGCCGGGGCGCGCCTGCCT180           
GCCCTGCTGGACGAGCAGCAGGTAAACGTGCTGCTCTACGACATGAACGGCTGTTACTCA240           
CGCCTCAAGG AGCTGGTGCCCACCCTGCCCCAGAACCGCAAGGTGAGCAAGGTGGAGATT300          
CTCCAGCACGTCATCGACTACATCAGGGACCTTCAGTTGGAGCTGAACTCGGAATCCGAA360           
GTTGGAACCCCCGGGGGCCGAGGGCTGCCGGTCCGCGCTCCGCTCAGCACCCT CAACGGC420          
GAGATCAGTGCCCTGACGGCCGAGGTGAGATCCAGATCCGACCACTAGATCATCCTTATA480           
CCGACGGGGAAACGGAGGCCAG502                                                 
(2) INFORMATION FOR SEQ ID NO:4:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
 (A) LENGTH: 148 amino acids                                              
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                   
MetLysValAlaSerGlySerThrAlaThrAlaAlaAlaGlyProThr                          
1 51015                                                                   
CysAlaLeuLysAlaGlyLysThrAlaSerGlyAlaGlyGluValVal                          
202530                                                                    
ArgCysL euSerGluGlnSerValAlaIleSerArgCysArgGlyAla                         
354045                                                                    
GlyAlaArgLeuProAlaLeuLeuAspGluGlnGlnValAsnValLeu                          
50 5560                                                                   
LeuTyrAspMetAsnGlyCysTyrSerArgLeuLysGluLeuValPro                          
65707580                                                                  
ThrLeuPro GlnAsnArgLysValSerLysValGluIleLeuGlnHis                         
859095                                                                    
ValIleAspTyrIleArgAspLeuGlnLeuGluLeuAsnSerGluSer                          
 100105110                                                                
GluValGlyThrProGlyGlyArgGlyLeuProValArgAlaProLeu                          
115120125                                                                 
SerThr LeuAsnGlyGluIleSerAlaLeuThrAlaGluValArgSer                         
130135140                                                                 
ArgSerAspHis                                                              
145                                                                       
(2) INFORMATION FOR SEQ ID NO:5:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 38 base pairs                                                 
 (B) TYPE: nucleic acid                                                   
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA                                                   
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                   
GATGTAGTCGATGACATGCTGCAGGATTTCCACCTTGC38                                  
(2) INFORMATION FOR SEQ ID NO:6:                                          
 (i) SEQUENCE CHARACTERISTICS:                                            
(A) LENGTH: 38 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA                                                   
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                   
GATGTAGTCTATTACACGCTGCAGGATCTCCACCTGGC 38                                 
(2) INFORMATION FOR SEQ ID NO:7:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 38 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA                                                   
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                   
CAGCTCCTTGA GGCGCGAGTAGCAGTCGTTCATGTCGT38                                 
(2) INFORMATION FOR SEQ ID NO:8:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 38 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA                                                   
(iii) HYPOTHETICAL: NO                                                    
( iv) ANTI-SENSE: NO                                                      
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                   
CAGTTCCCGCAGGCGTGAGTAGCAGCGGTTCATGTTGT38                                  
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