Abstract:
The present invention relates to a human PAB II gene containing transcribed polymorphic GCG repeat, which comprises a sequence as set forth in SEQ ID NO:18, which includes introns and flanking genomic sequence. Allelic variants of GCG repeat of the human PAB II gene are associated with a disease related with protein accumulation in the nucleus, such as polyalanine accumulation, or with swallowing difficulties, such as oculopharyngeal muscular dystrophy. The present invention also relates to a method for the diagnosis of a disease associated with protein accumulation in the nucleus, which comprises the steps of: a) obtaining a nucleic acid sample of a patient; and b) determining allelic variants of GCG repeat of the PAB II gene, and wherein long allelic variants are indicative of a disease related with protein accumulation in the nucleus.

Description:
RELATED APPLICATION(S) 
     This application is a continuation claiming priority to International Application No. PCT/CA98/01133, filed Dec. 7, 1998 (designating the U.S.), which claims priority to Canadian Patent No. 2,218,199, filed Dec. 9, 1997, the entire teachings of both of which are incorporated herein by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The invention relates to PAB II gene, and its uses thereof for the diagnosis, prognosis and treatment of a disease related with protein accumulation in nucleus, such as oculopharyngeal muscular dystrophy. 
     2. Description of Prior Art 
     Autosomal dominant oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease with a world-wide distribution. It usually presents itself in the sixth decade with progressive swallowing difficulties (dysphagia), eye lid drooping (ptosis) and and proximal limb weakness. Unique nuclear filament inclusions in skeletal muscle fibers are its pathological hallmark (Tome, F.M.S. &amp; Fardeau, Acta Neuropath. 49, 85-87 (1980)). Using the full power of linkage analysis in eleven French Canadian families, the oculopharyngeal muscular dystrophy gene was fine mapped on human chromosome 14 (Brais et al., 1997, Neuromuscular Disorders 7 (Suppl.1):S70-74). A region of 0.75 cM was thereby identified as a region containing the potential and unknown OMPD gene (Brais et al., 1997, supra). Unfortunately, the OMPD gene has yet to be isolated and its nucleic acid or protein sequence have yet to be described. 
     It would be highly desirable to be provided with a tool for the diagnosis, prognosis and treatment of a disease related with polyalanine accumulation in the nucleus, such as observed in oculopharyngeal muscular dystrophy. 
     SUMMARY OF THE INVENTION 
     One aim of the present invention is to provide a tool for the diagnosis, prognosis and treatment of a disease related with polyalanine accumulation in nucleus, such as oculopharyngeal muscular dystrophy. 
     Herein, the poly(A) binding protein II (PAB II) gene was isolated from a 217 kb candidate interval in chromosome 14q11. A (GCG)6 repeat encoding a polyalanine tract located at the N-terminus of the protein was expanded to (GCG)8-13 in the 144 OPMD families screened. More severe phenotypes were observed in compound heterozygotes for the (GCG)9 mutation and a (GCG)7 allele found in 2% of the population, whereas homozygosity for the (GCG)7 allele leads to autosomal recessive OPMD. Thus the (GCG)7 allele is an example of a polymorphism which can act as either a modifier of a dominant phenotype or as a recessive mutation. Pathological expansions of the polyalanine tract may cause mutated PAB II oligomers to accumulate as filament inclusions in nuclei. 
     In accordance with the present invention there is provided a human PAB II gene containing a transcribed polymorphic GCG repeat, which comprises a sequence as set forth in FIG. 4, which includes introns and flanking genomic sequence. 
     The allelic variants of GCG repeat of the human PAB II gene are associated with a disease related with protein accumulation in the nucleus, such as polyalanine accumulation, or with a disease related with swallowing difficulties, such as oculopharyngeal muscular dystrophy. 
     In accordance with the present invention there is also provided a method for the diagnosis of a disease associated with protein accumulation in the nucleus, which comprises the steps of: 
     a) obtaining a nucleic acid sample of said patient; and 
     b) determining allelic variants of a GCG repeat of the human PAB II gene; whereby long allelic variants are indicative of a disease related with protein accumulation in the nucleus, such as polyalanine accumulation and oculopharyngeal muscular dystrophy. 
     The long allelic variants have from about 245 to about 263 bp in length. 
     In accordance with the present invention there is also provided a non-human mammal model for the human PAB II gene, whose germ cells and somatic cells are modified to express at least one allelic variant of the PAB II gene and wherein said allelic variant of the PAB II is being introduced into the mammal, or an ancestor of the mammal, at an embryonic stage. 
     In accordance with the present invention there is also provided a method for the screening of therapeutic agents for the prevention and/or treatment of oculopharyngeal muscular dystrophy, which comprises the steps of: 
     a) administering the therapeutic agents to the non-human animal of the present invention or oculopharyngeal muscular dystrophy patients; and 
     b) evaluating the prevention and/or treatment of development of oculopharyngeal muscular dystrophy in this animal (such as a mammal) or in patients. 
     In accordance with the present invention there is also provided a method to identify genes, products thereof, or part thereof, which interact with a biochemical pathway affected by the PAB II gene, which comprises the steps of: 
     a) designing probes and/or primers using the PAB II gene and screening oculopharyngeal muscular dystrophy patients samples with said probes and/or primers; and 
     b) evaluating the role of the identified gene in oculopharyngeal muscular dystrophy patients. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIGS. 1A-1B illustrate the positional cloning of the PAB II gene; 
     FIGS. 2A-2G illustrate the OPMD (GCG)n expansion sizes and sequence of the mutation site (SEQ ID NO: 1nucleic acid mutation site; SEQ ID NO:2: nucleic acid mutation site without insertion; SEQ ID NO:3-9, nucleic acid mutation site with 1 through 7 (GCG) insertions, respectively; SEQ ID NO:10, amino acid sequence encoded by the nucleic acid mutation site; SEQ ID NO: 11-17, amino acid sequence encoded by the nucleic acid mutation site with 1 through 7 Alanine insertions, respectively); 
     FIG. 3 illustrates the age distribution of swallowing time (st) for French Canadian OPMD carriers of the (GCG)9 mutation; and 
     FIGS. 4A-4E illustrate the nucleotide sequence of human poly(A) binding protein II (hPAB II) (SEQ ID NO:18). 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     In order to identify the gene mutated in OPMD, a 350 kb cosmid contig was constructed between flanking markers D14S990 and D14S1457 (FIG.  1 A). Positions of the PAB II-selected cDNA clones were determined in relation to the EcoRI restriction map and the Genealogy-based Estimate of Historical Meiosis (GEHM)-derived candidate interval (Ronmmens, J. M. et al., in Proceedings of the third international workshop on the identification of transcribed sequences (eds. Hochgeschwender, U. &amp; Gardiner, K.) 65-79 (Plenum, New York, 1994)). 
     The human poly(A) binding protein II gene (PAB II) is encoded by the nucleotide sequence as set forth in FIG.  4 . 
     Twenty-five cDNAs were isolated by cDNA selection from the candidate interval (Rommens, J. M. et al., in Proceedings of the third international workshop on the identification of transcribed sequences (eds. Hochgeschwender, U. &amp; Gardiner, K.; 65-79; Plenum, New York, 1994). Three of these hybridized to a common 20 kb EcoRI restriction fragment and showed high sequence homology to the bovine poly(A) binding protein II gene(bPAB II) (FIG.  1 A). The PAB II gene appeared to be a good candidate for OPMD because it mapped to the genetically defined 0.26 cM candidate interval in 14q11 (FIG.  1 A), its mRNA showed a high level of expression in skeletal muscle, and the PAB II protein is exclusively localized to the nucleus (Krause, S. et al., Exp. Cell Res. 214, 75-82 (1994)) where it acts as a factor in mRNA polyadenylation (Whale, E., Cell 66, 759-768 (1991); Whale, E. et al., J. Biol. Chem. 268, 2937-2945 (1993); Bienroth, S. et al., EMBO J. 12, 585-594 (1993)). 
     A 8 kb HindIII genomic fragment containing the PAB II gene was subcloned and sequenced (6002 bp; GenBank: AF026029) (Nemeth, A. et al., Nucleic Acids Res. 23, 4034-4041 (1995)) (FIG.  1 B). Genomic structure of the PAB II gene, and position of the OPMD (GCG)n expansions. Exons are numbered. Introns 1 and 6 are variably present in 60% of cDNA clones. ORF, open reading frame; cen, centromere and tel, telomere. 
     The coding sequence was based on the previously published bovine sequence (GenBank: X89969) and the sequence of 31 human cDNAs and ESTs. The gene is composed of 7 exons and is transcribed in the cen-qter orientation (FIG.  1 B). Multiple splice variants are found in ESTs and on Northern blots (Nemeth, A. et al., Nucleic Acids Res. 23, 4034-4041 (1995)). In particular, introns 1 and 6 are present in more than 60% of clones (FIG.  1 B)(Nemeth, A. et al., Nucleic Acids Res. 23, 4034-4041 (1995)). The coding and protein sequences are highly conserved between human, bovine and mouse (GenBank: U93050). 93% of the PAB II sequence was readily amenable to RT-PCR- or genomic-SSCP screening. No mutations were uncovered using both techniques. However, a 400 bp region of exon 1 containing the start codon could not be readily amplified. This region is 80% GC rich. It includes a (GCG)6 repeat which codes for the first six alanines of a homopolymeric stretch of 10 (FIG.  2 G). Nucleotide sequence of the mutated region of PAB II as well as the amino acid sequences of the N-terminus polyalanine stretch and position of the OPMD alanine insertions is also shown in FIG.  2 . 
     Special conditions were designed to amplify by PCR a 242 bp genomic fragment including this GCG-repeat. The (GCG)6 allele was found in 98% of French Canadian non-OPMD control chromosomes, whereas 2% of chromosomes carried a (GCG)7 polymorphism (n=86) (Brais, B. et al., Hum. Mol. Genet. 4, 429-434 (1995)). 
     Screening OPMD cases belonging to 144 families showed in all cases a PCR product larger by 6 to 21 bp than that found in controls (FIG.  2 A). (GCG)6 normal allele (N) and the six different (GCG)n expansions observed in 144 families. 
     Sequencing of these fragments revealed that the increased sizes were due to expansions of the GCG repeat (FIG.  2 G). FIG. 2F shows the sequence of the (GCG)9 French Canadian expansion in a heterozygous parent and his homozygous child. Partial sequence of exon 1 in a normal (GCG)6 control (N), a heterozygote (ht.) and a homozygote (hm.) for the (GCG)9-repeat mutation. The number of families sharing the different (GCG)n-repeats expansions is shown in Table 1. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Number of families sharing the different 
               
               
                 dominant (GCG)n OPMD mutations 
               
             
          
           
               
                   
                 Mutations 
                 Polyalanine† 
                 Families 
               
               
                   
                   
               
             
          
           
               
                   
                 (GCG)8 
                 12 
                 4 
               
               
                   
                 (GCG)9 
                 13 
                 99 
               
               
                   
                 (GCG)10 
                 14 
                 19 
               
               
                   
                 (GCG)11 
                 15 
                 16 
               
               
                   
                 (GCG)12 
                 16 
                 5 
               
               
                   
                 (GCG)13 
                 17 
                 1 
               
               
                   
                 Total 
                   
                 144 
               
               
                   
                   
               
               
                   
                 †, 10 alanine residues in normal PAB II.  
               
             
          
         
       
     
     The (GCG)9 expansion shared by 70 French Canadian families is the most frequent mutation we observed (Table 1) The (GCG)9 expansion is quite stable, with a single doubling observed in family F151 in an estimated 598 French Canadian meioses (FIG.  2 C). The doubling of the French Canadian (GCG)9 expansion is demonstrated in Family F151. 
     This contrasts with the unstable nature of previously described disease-causing triplet repeats (Rosenberg, R. N., New Eng. J. Med. 335, 1222-1224 (1996)). 
     Genotyping of all the participants in the clinical study of French Canadian OPMD provided molecular insights into the clinical variability observed in this condition. The genotypes for both copies of the PAB II mutated region were added to an anonymous version of this clinical database of 176 (GCG)9 mutation carriers (Brais, B. et al., Hum. Mol. Genet. 4, 429-434 (1995)). Severity of the phenotype can be assessed by the swallowing time (st) in seconds taken to drink 80 cc of ice-cold water (Brais, B. et al., Hum. Mol. Genet. 4, 429-434 (1995); Bouchard, J.-P. et al., Can. J. Neurol. Sci. 19, 296-297 (1992)). The late onset and progressive nature of the muscular dystrophy is clearly illustrated in heterozygous carriers of the (GCG)9 mutation (bold curve in FIG. 3) when compared to the average st of control (GCG)6 homozygous participants(n=76, thinner line in FIG.  3 ). The bold curve represents the average OPMD st for carriers of only one copy of the (GCG)9 mutation (n=169), while the thinner line corresponds to the average st for (GCG)6 homozygous normal controls (n=76). The black dot corresponds to the st value for individual VIII. Roman numerals refer to individual cases shown in FIGS. 2B,  2 D and discussed in the text. The genotype of a homozygous (GCG)9 patient and her parents is shown in FIG.  2 B. Independent segregation of the (GCG)7 allele is also shown. Of note, case V has a more severe OPMD phenotype (FIG.  2 D). 
     Two groups of genotypically distinct OPMD cases have more severe swallowing difficulties. Individuals I, II, and III have an early-onset disease and are homozygous for the (GCG)9 expansion (P&lt;10−5) (FIGS. 2B, F). Cases IV, V, VI and VII have more severe phenotypes and are compound heterozygotes for the (GCG)9 mutation and the (GCG)7 polymorphism (P&lt;10−5). In FIG. 2D the independent segregation of the two alleles is shown. Case V, who inherited the French Canadian (GCG)9 mutation and the (GCG)7 polymorphism, is more symptomatic than his brother VIII who carries the (GCG)9 mutation and a normal (GCG)6 allele (FIGS.  2 D and  3 ). The (GCG)7 polymorphism thus appears to be a modifier of severity of dominant OPMD. Furthermore, the (GCG)7 allele can act as a recessive mutation. This was documented in the French patient IX who inherited two copies of the (GCG)7 polymorphism and has a late-onset autosomal recessive form of OPMD (FIG.  2 E). Case IX, who has a recessive form of OPMD, is shown to have inherited two copies of the (GCG)7 polymorphism. 
     This is the first description of short trinucleotide repeat expansions causing a human disease. The addition of only two GCG repeats is sufficient to cause dominant OPMD. OPMD expansions do not share the cardinal features of “dynamic mutations”. The GCG expansions are not only short they are also meiotically quite stable. Furthermore, there is a clear cut-off between the normal and abnormal alleles, a single GCG expansion causing a recessive phenotype. The PAB II (GCG)7 allele is the first example of a relatively frequent allele which can act as either a modifier of a dominant phenotype or as a recessive mutation. This dosage effect is reminiscent of the one observed in a homozygote for two dominant synpolydactyly mutations. In this case, the patient had more severe deformities because she inherited two duplications causing an expansion in the polyalanine tract of the HOXD13 protein (Akarsu, A. N. et al., Hum. Mol. Genet. 5, 945-952 (1996)). A duplication causing a similar polyalanine expansion in the a subunit 1 gene of the core-binding transcription factor (CBF(1) has also been found to cause dominant cleido-cranial dysplasia (Mundlos, S. et al., Cell 89, 773-779 (1997)). The mutations in these two rare diseases are not triplet repeats. The are duplications of “cryptic repeats” composed of mixed synonymous codons and are thought to result from unequal crossing over (Warren, S. T., Science 275, 408-409 (1997)). In the case of OPMD, slippage during replication causing a reiteration of the GCG codon is a more likely mechanism (Wells, D. R., J. Biol. Chem. 271, 2875-2878 (1996)). 
     Different observations converge to suggest that a gain of function of PAB II may cause the accumulation of nuclear filaments observed in OPMD (Tome, F. M. S. &amp; Fardeau, Acta Neuropath. 49, 85-87 (1980)). PAB II is found mostly in dimeric and oligomeric forms (Nemeth, A. et al., Nucleic Acids Res. 23, 4034-4041 (1995)). It is possible that the polyalanine tract plays a role in polymerization. Polyalanine stretches have been found in many other nuclear proteins such as the HOX proteins, but their function is still unknown (Davies, S. W. et al., Cell 90, 537-548 (1997)). Alanine is a highly hydrophobic amino acid present in the cores of proteins. In dragline spider silk, polyalanine stretches are thought to form P-sheet structures important in ensuring the fibers&#39; strength (Simmons, A. H. et al., Science 271, 84-87 (1996)). Polyalanine oligomers have also been shown to be extremely resistant to chemical denaturation and enzymatic degradation (Forood, B. et al., Bioch. and Biophy. Res. Corn. 211, 7-13 (1995)). One can speculate that PAB II oligomers comprised of a sufficient number of mutated molecules might accumulate in the nuclei by forming undegradable polyalanine rich macromolecules. The rate of the accumulation would then depend on the ratio of mutated to non-mutated protein. The more severe phenotypes observed in homozygotes for the (GCG)9 mutations and compound heterozygotes for the (GCG)9 mutation and (GCG)7 allele may correspond to the fact that in these cases PAB II oligomers are composed only of mutated proteins. The ensuing faster filament accumulation could cause accelerated cell death. The recent description of nuclear filament inclusions in Huntington&#39;s disease, raises the possibility that “nuclear toxicity” caused by the accumulation of mutated homopolymeric domains is involved in the molecular pathophysiology of other triplet repeats diseases (Davies, S. W. et al., Cell 90, 537-548 (1997); Scherzinger, E. et al., Cell 90, 549-558 (1997); DiFiglia, M. et al., Science 277, 1990-1993 (1997)). Future immunocytochemical and expression studies will be able to test this pathophysiological hypothesis and provide some insight into why certain muscle groups are more affected while all tissues express PAB II. 
     Methods 
     Contig and cDNA Selection 
     The cosmid contig was constructed by standard cosmid walking techniques using a gridded chromosome 14specific cosmid library (Evans, G. A. et al., Gene 79, 9-20 (1989)). The cDNA clones were isolated by cDNA selection as previously described (Rommens, J. M. et al., in Proceedings of the third international workshop on the identification of transcribed sequences (eds. Hochgeschwender, U. &amp; Gardiner, K.) 65-79 (Plenum, New York, 1994)). 
     Three cDNA clones corresponding to PAB II were sequenced (Sequenase, USB). Clones were verified to map to cosmids by Southern hybridization. The 8 kb HindIII restriction fragment was subcloned from cosmid 166G8 into pBluescriptII (SK) (Stratagene). The clone was sequenced using primers derived from the bPABII gene and human EST sequences. Sequencing of the PAB II introns was done by primer walking. 
     PAB II Mutation Screening and Sequencing 
     All cases were diagnosed as having OPMD on clinical grounds (Brais, D. et al., Hum. Mol. Genet. 4, 429-434 (1995)). RT-PCR and genomic SSCP analyses were done using standard protocols (Lafrenière, R. G. et al., Nat. Genet. 15, 298-302 (1997)). The primers used to amplify the PAB II mutated region were: 5′-CGCAGTGCCCCGCCTTAGA-3′ (SEQ ID NO:4) and 5′-ACAAGATGGCGCCGCCGCCCCGGC-3′ (SEQ ID NO:5). PCR reactions were performed in a total volume of 15 μl containing: 40 ng of genomic DNA; 1.5 μg of BSA; 1 μM of each primer; 250 μM dCTP and dTTP; 25 μM dATP; 125 μM of dGTP and 125 μM of 7-deaza-dGTP (Pharmacia); 7.5% DMSO; 3.75 μCi[ 35 S]dATP, 1.5 unit of Taq DNA polymerase and 1.5 mM MgC12 (Perkin Elmer). For non-radioactive PCR reactions the [ 35 S]dATP was replaced by 225 μM of dATP. The amplification procedure consisted of an initial denaturation step at 95° C. for five minutes, followed by 35 cycles of denaturation at 95° C. for 15 s, annealing at 70° C. for 30 s, elongation at 74° C. for 30 s and a final elongation at 74° C. for 7 min. Samples were loaded on 5% polyacrylamide denaturing gels. Following electrophoresis, gels were dried and autoradiographs were obtained. Sizes of the inserts were determined by comparing to a standard M13 sequence (Sequenase™, USB). Fragments used for sequencing were gel-purified. Sequencing of the mutated fragment using the Amplicycle kit™(Perkin Elmer) was done with the 5′-CGCAGTGCCCCGCCTTAGAGGTG-3′ (SEQ ID NO:6) primer at an elongation temperature of 68° C. 
     Stability of (GCG)-repeat Expansions 
     The meiotic stability of the (GCG)9-repeat was estimated based on a large French Canadian OPMD cohort. It had been previously established that a single ancestral OPMD carrier chromosome was introduced in the French Canadian population by three sisters in 1648. Seventy of the seventy one French Canadian OPMD families tested to date segregate a (GCG)9 expansion. However, in family F151, the affected brother and sister, despite sharing the French Canadian ancestral haplotype, carry a (GCG)12 expansion, twice the size of the ancestral (GCG)9 mutation (FIG.  2 C). In this founder effect study, it is estimated that 450 (304-594) historical meioses shaped the 123 OPMD cases belonging to 42 of the 71 enrolled families. The screening of the full set of participants allowed an identification of another 148 (GCG)9 carrier chromosomes. Therefore, it is estimated that a single mutation of the (GCG)9 expansion has occurred in 598 (452-742) meioses. 
     Genotype-phenotype Correlations 
     176 carriers of at least one copy of the (GCG)9 mutation were examined during the early stage of the linkage study. All were asked to swallow 80 cc of ice-cold water as rapidly as possible. Testing was stopped after 60 seconds. The swallowing time (st) was validated as a sensitive test to identify OPMD cases (Brais, B. et al., Hum. Mol. Genet. 4, 429-434 (1995); Bouchard, J.-P. et al., Can. J. Neurol. Sci. 19, 296-297 (1992)). The st values for 76 (GCG)6 homozygotes normal controls is illustrated in FIG.  3 . Analyses of variance were computed by two-way ANOVA (SYSTAT package). For the (GCG)9 homozygotes their mean st value was compared to the mean value for all (GCG)9 heterozygotes aged 35-40 (P&lt;10 −5 ). For the (GCG)9 and (GCG)7 compound heterozygotes their mean st value was compared to the mean value for all (GCG)9 heterozygotes aged 45-65 (P&lt;10 −5 ). 
     While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims. 
     
       
         
           
             21 
           
           
             1 
             30 
             DNA 
             Homo sapiens 
           
            1
atggcggcgg cggcggcggc ggcagcagca                                      30
 
           
             2 
             24 
             DNA 
             Homo sapiens 
           
            2
atggcggcgg cggcggcggc ggca                                            24
 
           
             3 
             27 
             DNA 
             Homo sapiens 
           
            3
atggcggcgg cggcggcggc ggcggca                                         27
 
           
             4 
             30 
             DNA 
             Homo sapiens 
           
            4
atggcggcgg cggcggcggc ggcggcggca                                      30
 
           
             5 
             33 
             DNA 
             Homo sapiens 
           
            5
atggcggcgg cggcggcggc ggcggcggcg gca                                  33
 
           
             6 
             36 
             DNA 
             Homo sapiens 
           
            6
atggcggcgg cggcggcggc ggcggcggcg gcggca                               36
 
           
             7 
             39 
             DNA 
             Homo sapiens 
           
            7
atggcggcgg cggcggcggc ggcggcggcg gcggcggca                            39
 
           
             8 
             42 
             DNA 
             Homo sapiens 
           
            8
atggcggcgg cggcggcggc ggcggcggcg gcggcggcgg ca                        42
 
           
             9 
             45 
             DNA 
             Homo sapiens 
           
            9
atggcggcgg cggcggcggc ggcggcggcg gcggcggcgg cggca                     45
 
           
             10 
             19 
             PRT 
             Homo sapiens 
           
            10
Met Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Ala Ala Gly Gly
 1               5                  10                  15
Arg Gly Ser
 
           
             11 
             16 
             PRT 
             Homo sapiens 
           
            11
Met Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Ala Ala Gly
 1               5                  10                  15
 
           
             12 
             17 
             PRT 
             Homo sapiens 
           
            12
Met Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Ala Ala
 1               5                  10                  15
Gly
 
           
             13 
             18 
             PRT 
             Homo sapiens 
           
            13
Met Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Ala
 1               5                  10                  15
Ala Gly
 
           
             14 
             19 
             PRT 
             Homo sapiens 
           
            14
Met Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly
 1               5                  10                  15
Ala Ala Gly
 
           
             15 
             20 
             PRT 
             Homo sapiens 
           
            15
Met Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
 1               5                  10                  15
Gly Ala Ala Gly
            20
 
           
             16 
             21 
             PRT 
             Homo sapiens 
           
            16
Met Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
 1               5                  10                  15
Ala Gly Ala Ala Gly
            20
 
           
             17 
             22 
             PRT 
             Homo sapiens 
           
            17
Met Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
 1               5                  10                  15
Ala Ala Gly Ala Ala Gly
            20
 
           
             18 
             6002 
             DNA 
             Homo sapiens 
             
               misc_feature 
               (4616)...(4616) 
               n= A,T,C, or G 
             
           
            18
aatgaaggtg gacacccaaa tagccccaat acaaatgcct gttcaatcaa ccaaacatct     60
aagcagcaca tctatgtggt agcatattgc caggccgtga gactgcgaat ataaatagga    120
accgcccctc atctgcaggc gctcacaacc tagttagcaa acagtaaaac aattaagcgc    180
gccgtggaca taggcccact tgtcctggga aatgagggga agctggggtt tgcagtggtt    240
tgattgaagg gggactacat gttagaggca cagactgggt gcaggtacac ccaaaggaac    300
gagaagagtg gaaggaaaca acatccacaa agtaaccaca tgctggcgta tcgaaggccg    360
tgatttacgg ttttgagact ttacctcgcc agcaaagggg ggccagtctg ttagcggtgc    420
agattggagg ggtgacattg gaagctgtcc aggaaaaaga aaatggaact ggggagcaga    480
aggcctacgc aagagggcgg gacagacagg acttgtgact agtagctctg gactgaggaa    540
tcctccctgc tttctggtgc gggagagcta gtggatgatg gtgccaataa cctggatggg    600
gaaagtaagc tccctcctgg aatgcttcat tcacaacctc cattttcagc aacatcccat    660
ctactggtgc ttcctggtcg agatacaagt ttcctgaaac tgctgctctg ttttgggcct    720
cacccggcca acagctcact agctggcaag cagtagtatc aagatggcgg ccccctagga    780
ctggctagtc atgtgacctc gggtttccca agtttgaagc ccggcagtcc tttcgggggc    840
aaggttcacc tgtcacgaaa cgagtgtcac cccttcgact ctcgcaagcc aatcggcatc    900
tgagactggg ccactgcggt gaggcgatcg gaagattggt cctttccagt cgcctagcta    960
gggccaatca cggagcgtcc catacttcgc gggcccgccc gtaggccggg gagaagcagg   1020
aatatcgtca cagcgtggcg gtattattac ctaaggactc gataggaggt gggacgcgtg   1080
ttgattgaca ggcagatttc cctaccggga tttgagaatt tggcgcagtg cccgccttag   1140
aggtgcgctt atttgattgc caagtaatat tccccaatgg agtactagct catggtgacg   1200
ggcaggcagc ttgagctaat gagtcctccg tggccggcgc agctctccac atgccgggcg   1260
gcgggcccca gtctgagcgg cgatggcggc ggcggcggcg gcggcagcag cagcgggggc   1320
tgcgggcggt cggggctccg ggccggggcg gcggcgccat cttgtgcccg gggccggtgg   1380
ggaggccggg gagggggccc cggggggcgc aggggactac gggaacggcc tggagtctga   1440
ggaactggag cctgaggagc tgctgctgga gcccgagccg gagcccgagc ccgaagagga   1500
gccgccccgg ccccgcgccc ccccgggagc tccgggccct gggcctggtt cgggagcccc   1560
cggcagccaa gaggaggagg aggagccggg actggtcgag ggtgacccgg gggacggcgc   1620
cattgaggac ccggtgagga aggagggcga gcgagcaggc cggcggctgg cgcgtcactg   1680
gaggcccaga gctcgggcga gcggtggcag gcggggggtg gggttgggcg gggaataacg   1740
tggctggggc gggtcgggcc ggggatgggt cagcgatcac tacaaggggc ccgactggct   1800
tgattcgggc gtcacgggtg cctagtgttg ttctagagag ggtagctttt cttttatcac   1860
gaccctcgca tggggcgagg gaaatggccg agcatggctg aggcgcgctc tggccgagag   1920
cagggcacag cccctgcgtt ggttcctctt aagctgtcct ccataccctc cccacttata   1980
ttaggagctg gaagctatca aagctcgagt cagggagatg gaggaagaag ctgagaagct   2040
aaaggagcta cagaacgagg tagagaagca gatgaatatg agtccacctc caggcaatgc   2100
tgagtaactg gcggttgcac gcggagcccg ggttctcggg ttggaagggt tgtggggagg   2160
atggggaatg tggggttaga tactcggcac cctggagctg cttgtctgag ctattatgac   2220
tgtgccgcgg tcatagtccg ttgtgtgttc ctctgacctt tgtgaggcag aactgatatt   2280
ttggtggtgg tagccttgtg cctccctttg tcctgttata attgtgttgc tctttattct   2340
tagtctacgt ctatctttct ttggtagagg ttgcgtgctc gcatttgacc ttcaaatcta   2400
atagtttttc ctccaattgg agacgcttta ggattctaag agaaagcaag ctggaagggg   2460
tttccccttt aaattctaga aatgtggagt ctcagcccac ttaattttgc tcactcttaa   2520
aagcatttca accaaagcca ttcattaggg atttgatttg gagggcagga gggattccta   2580
tactgtttta agtgtgtatt aattctttca atttatcgaa ttatttagtg agtaacctgc   2640
tatgcactag gcactattct cggcttgtgg gtacagcagg gaacagcaca gaccaaaatc   2700
tttgccttca ctgagcttat gggatagtgc tggtggtgga agtgcaacat attggtcaag   2760
tagaaaacaa gtgtgtggtt tttgtaaaaa attatttttt cctgatagct ggcccggtga   2820
tcatgtccat tgaggagaag atggaggctg atgcccgttc catctatgtt ggcaatgtga   2880
cgtactgggg ctctgactgg ggttgggggc aagttcttct tttggggaat tatttaatag   2940
tcctgaaaga acatctccgg gatagatgtg gttttgggtg tggagggagt gtgggaagga   3000
ggttaaaggt aatggaatga tcagtaatca gcaaaggctc tgggtttgga aggaaaagag   3060
attaattcct caaattacca gatttcatgt gctttggtgt atgatggccc agaccaaagg   3120
ctcgggaggg ttcttttgag acaggaattt gcctggtgcc tgtgaaattt ttctcctctc   3180
atcaggtgga ctatggtgca acagcagaag agctggaagc tcactttcat ggctgtggtt   3240
cagtcaaccg tgttaccata ctgtgtgaca aatttagtgg ccatcccaaa ggtaaagtaa   3300
aggggagtaa gttgagataa tttaaattac agtgtacaaa tagataaatt atgttttata   3360
ttgagcagta agttatttgg tgttaacaca ggtgatctgt gtcatttaag atcatggcat   3420
taatgttgat atatcaggag ttgcacctaa atgtcttcag aggccagata acaaaaatga   3480
aggctagatg tgggtgggat tacgaactag aaggggaggg gcagcttcta cttggcctat   3540
tatggcatat ggaaattcag gccctgtgtg tcttattttt acaaatttca aagagtagct   3600
ggaaatttta aaatttaaat gatttcgaat gattgaaatt ttccatttag aagaattttg   3660
acaaataaaa aatataactg cattgtagcc caaaacgaag catgcctgca ggttgaattt   3720
gacctgtgag gtatttgtaa cctcagagag atacaatgac aattcttttc aggtttgcgt   3780
atatagagtt ctcagacaaa gagtcagtga ggacttcctt ggccttagat gagtccctat   3840
ttagaggaag gcaaatcaag gtaagcctat gtccattgct gttctagttg tgtataaact   3900
ctccaggttg cctttaaggc tatcatttgt tcatctctga ctcaggtgat cccaaaacga   3960
accaacagac caggcatcag cacaacagac cggggttttc cacgagcccg ctaccgcgcc   4020
cggaccacca actacaacag ctcccgctct cgattctaca gtggttttaa cagcaggccc   4080
cggggtcgcg tctacaggtc aggatagatg ggctgctcct ctttcccccg cctcccgtga   4140
gccccgtatg cttcctcctc tctggtctga ggaacctccc tccccccacc cctccccgtg   4200
gtcttcagga actttgtctc ctgcctgtgc aggttgagga aggtagttgc aggccaggcc   4260
agaaggcagc ctcatcatct tttctgcagt agaaattggt gataagggct gcatccctcc   4320
cttggttcaa agaggcttcc acccccagcc ttttttttct tgggagttgg tggcatttga   4380
aggtgtttgc ggacaaaact gggaggaaca gggcctccag gaagttgaaa gcactgcttg   4440
gacatttgtt acttttttcg gagttaggga gggattgaag actgaacctc ccttggaaga   4500
ataccagagg ctagctagtt gatcctccca acagccttgt gggaggattt tgagatactt   4560
attctttatt tgagccagtc ttgcaaggtt aacttctcac tgggcctagt gtggtnccca   4620
ggtttttgcc ttgcttcact tctgtctcta catttaaata gacgggttag gcatataaac   4680
cttggctttt cataagctct acctgcctat ccccaggagt tagggaggat ctatttgtga   4740
aggccctagg gtttaaaaac tgtggaggac tgaaaaactg gataaaaagg gggtcctttt   4800
ccttgcccct gtctctcact cagatgcgct tctttttcgc cactgtttgg caaagttttc   4860
tgttaagccc ccctccccct gccccagttc tcccaggtgc gttactattt ctgggatcat   4920
ggggtcggtt ttaggacact tgaacacttc ttttcccccc ttcccttcac agtaactggg   4980
gcaggggcct acggggaggg gcttgtactg aactatctag tgatcacgtt aacacctaac   5040
tctccttctt tcttccaggg gccgggctag agcgacatca tggtattccc cttactaaaa   5100
aaagtgtgta ttaggaggag agagaggaaa aaaagaggaa agaaggaaaa aaaaaagaat   5160
taaaaaaaaa aaaaagaaaa acagaagatg accttgatgg aaaaaaaata ttttttaaaa   5220
aaaagatata ctgtggaagg ggggagaatc ccataactaa ctgctgagga gggacctgct   5280
ttggggagta ggggaaggcc cagggagtgg ggcagggggc tgcttattca ctctggggat   5340
tcgccatgga cacgtctcaa ctgcgcaagc tgcttgccca tgtttccctg cccccttcac   5400
ccccttgggc ctgctcaagg gtaggtgggc gtgggtggta ggagggtttt ttttacccag   5460
ggctctggaa ggacaccaaa ctgttctgct tgttaccttc cctcccgtct tctcctcgcc   5520
tttcacagtc ccctcctgcc tgctcctgtc cagccaggtc taccacccac cccacccctc   5580
tttctccggc tccctgcccc tccagattgc ctggtgatct attttgtttc cttttgtgtt   5640
tctttttctg ttttgagtgt ctttctttgc aggtttctgt agccggaaga tctccgttcc   5700
gctcccagcg gctccagtgt aaattcccct tccccctggg gaaatgcact accttgtttt   5760
ggggggttta ggggtgtttt tgtttttcag ttgttttgtt tttttgtttt ttttttttcc   5820
tttgcctttt ttccctttta tttggaggga atgggaggaa gtgggaacag ggaggtggga   5880
ggtggatttt gtttattttt ttagctcatt tccaggggtg ggaatttttt tttaatatgt   5940
gtcatgaata aagttgtttt tgaaaataaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa   6000
aa                                                                  6002
 
           
             19 
             19 
             DNA 
             Artificial Sequence 
             
               oligonucleotide 
             
           
            19
cgcagtgccc cgccttaga                                                  19
 
           
             20 
             24 
             DNA 
             Artificial Sequence 
             
               oligonucleotide 
             
           
            20
acaagatggc gccgccgccc cggc                                            24
 
           
             21 
             23 
             DNA 
             Artificial Sequence 
             
               oligonucleotide 
             
           
            21
cgcagtgccc cgccttagag gtg                                             23